Novel Carbohydrate Profile Compositions From Human Cells and Methods for Analysis and Modification Thereof

ABSTRACT

The invention describes methods for production of novel composition of glycans, glycomes, from human multipotent stem cells. The invention is further directed to methods for modifying the glycomes and analysis of the glycomes and the modified glycomes. Furthermore the invention is directed to stem cells carrying the modified glycomes on their surfaces.

FIELD OF THE INVENTION

The invention describes methods for production of novel composition ofglycans, glycomes, from human multipotent stem cells. The invention isfurther directed to methods for modifying the glycomes and analysis ofthe glycomes and the modified glycomes. Furthermore the invention isdirected to stem cells carrying the modified glycomes on their surfaces.

The glycomes are preferably analysed by profiling methods able to detectreproducibly and quantitatively numerous individual glycan structures atthe same time. The most preferred type of the profile is a massspectrometric profile. The invention further describes uses of themethods for analytics and diagnostics. The methods are especiallydirected to analysis of glycan profiles from multipotent stem cells andeffects of various reagents having effect on cell glycosylation. Thepresent invention is specifically directed to analysis of specifiedN-glycan and O-glycan structure types as markers of the stem cells andfurther to uses of the analysed structures.

BACKGROUND OF THE INVENTION

Numerous methods have been developed for analysis of glycan structuresmainly from purified proteins. These methods describe generaltechnologies of N-glycan and O-glycan release, purification and analysisof the products by various methods including mass spectrometry. Usuallyexact analysis of material has required purification of specific glycansand numerous chemical and analytic methods.

The background further includes comparison of individual specific N- andO-glycans from healthy tissue and tissue affected by a disease. Thesemethods do not show the possibility to produce mass spectrometricprofiles, or quantitative data that allows comparison between samplescomprising numerous components. The special purification methods of thepresent invention have not been described previously.

Molecular profiling methods have been described for proteins, peptides,and nucleic acids. Some of these methods use small tissue samples. Theanalytic conditions and sensitivity for protein and nucleic acidanalytics is however very different from glycan sample analysis.

The present invention describes methods for production of free glycanmixtures from human stem cells. The novel method reveals a broad rangeof glycan structures observable by the novel analysis methods revealingnumerous novel characteristic of special quantitative cell derivedglycan compositions. The range of glycans from materials, whichglycosylation is largely unknown, reveals large amount of usefulinformation about the status. The invention shows effective very lowscale purification methods allowing separation of glycans from variousother cellular components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Example of glycan signal analysis of MALDI-TOF massspectrometric data. A. Mass spectrometric raw data showing a window ofneutral N-glycan mass spectrum in positive ion mode, B. Glycan profilegenerated from the data in A.

FIG. 2. Example of glycan signal analysis of MALDI-TOF massspectrometric data. A. Mass spectrometric raw data showing a window ofsialylated N-glycan mass spectrum in negative ion mode, B. Glycanprofile generated from the data in A.

FIG. 3. α2,3-sialidase profiling analysis of cord blood CD133+ andCD133− cells. Sialylated glycan fractions isolated after the reaction,showing the sialylated N-glycans bearing sialic acid residues resistantto the action of α2,3-sialidase. Light columns: CD133+ cells; darkcolumns: CD133− cells.

FIG. 4. α2,3-sialidase profiling analysis of cord blood CD133+ andCD133− cells. Neutral glycan fractions isolated after the reaction,showing the N-glycan core sequences of sialylated N-glycans that bearedonly α2,3-sialidase sensitive sialic acid residues. Light columns:CD133+ cells; dark columns: CD133− cells.

FIG. 5. α2,3-sialidase analysis of sialylated N-glycans isolated from A.cord blood CD133⁺ cells and B. CD133⁻ cells. The columns represent therelative proportions of a monosialylated glycan signal at m/z 2076 (SA₁)and the corresponding disialylated glycan signal at m/z 2367 (SA₂), asdescribed in the text. In cord blood CD133⁻ cells, the relativeproportions of the SA₁ and SA₂ glycans do not change markedly uponα2,3-sialidase treatment (B), whereas in CD133⁺ cells the proportion ofα2,3-sialidase resistant SA₂ glycans is significantly smaller thanα2,3-sialidase resistant SA₁ glycans (A).

FIG. 6. Neutral N-glycan profiles of a cord blood mononuclear cellpopulation.

FIG. 7. Sialylated N-glycan profiles of a cord blood mononuclear cellpopulation.

FIG. 8. Profiles of combined neutral and sialylated N-glycan fiactionsof a cord blood mononuclear cell population, after broad-rangeneuraminidase treatment of the sialylated fraction.

FIG. 9. Neutral N-glycan profiles of two cord blood derived mesenchymalstem cell lines. Light columns: cell line 1; dark columns: cell line 2.

FIG. 10. Sialylated N-glycan profiles of two cord blood derivedmesenchymal stem cell lines. Light columns: cell line 1; dark columns:cell line 2.

FIG. 11. Neutral N-glycan profiles of a cord blood derived mesenchymalstem cell line and cells differentiated into adipogenic direction. Lightcolumns: mesenchymal stem cell line; dark columns: mesenchymal stem cellline in adipogenic medium.

FIG. 12. Neutral N-glycan profiles of a cord blood derived mesenchymalstem cell line before (light columns) and after (dark columns)α-mannosidase digestion.

FIG. 13. Neutral N-glycan profiles of a cord blood derived mesenchymalstem cell line before (light columns) and after (dark columns)β1,4-galactosidase digestion.

FIG. 14. Neutral N-glycan profiles of a cord blood derived mesenchymalstem cell line, grown in adipogenic medium, before (light columns) andafter (dark columns) β1,4-galactosidase digestion.

FIG. 15. Neutral N-glycan profiles of a bone marrow derived mesenchymalstem cell line and cells differentiated into osteogenic direction. Lightcolumns: mesenchymal stem cell line in proliferation medium; darkcolumns: mesenchymal stem cell line in osteogenic medium.

FIG. 16. Sialylated N-glycan profiles of a bone marrow derivedmesenchymal stem cell line and cells differentiated into osteogenicdirection. Light columns: mesenchymal stem cell line in proliferationmedium; dark columns: mesenchymal stem cell line in osteogenic medium.

FIG. 17. Profiles of combined neutral and sialylated N-glycan fractionsof a bone marrow derived mesenchymal stem cell line and cellsdifferentiated into osteogenic direction, after broad-rangeneuraminidase treatment of the sialylated fraction. Light columns:mesenchymal stem cell line in proliferation medium; dark columns:mesenchymal stem cell line in osteogenic medium.

FIG. 18. Neutral N-glycan profiles of a human embryonic stem cell line(light columns), cells differentiated into embryoid bodies (darkcolumns), and st3 differentiated cells (blank columns).

FIG. 19. Sialylated N-glycan profiles of a human embryonic stem cellline (light columns), cells differentiated into embryoid bodies (darkcolumns), and st.3 differentiated cells (blank columns).

FIG. 20. Neutral N-glycan profiles of four human embryonic stem celllines (differently shaded columns, BESC lines 1-4).

FIG. 21. Sialylated N-glycan profiles of four human embryonic stem celllines (differently shaded columns, hESC lines 1-4).

FIG. 22. Sialylated N-glycan profiles of two human fibroblast feedercell samples: Light columns: cells grown separately from stem cells;dark columns: cells grown together with stem cells (feeder layer cells).

FIG. 23. Cord blood mononuclear cell sialylated N-glycan profiles before(light columns) and after (dark columns) subsequent broad-rangesialidase and α2,3-sialyltransferase reactions. The m/z values refer toTable 16.

FIG. 24. Cord blood mononuclear cell sialylated N-glycan profiles before(light columns) and after (dark columns) subsequentα2,3-sialyltransferase and α1,3-fucosyltransferase reactions. The m/zvalues refer to Table 16.

FIG. 25. Sialylated N-glycan profiles of human fibroblast feeder cells(light columns) and mouse fibroblast feeder cells (dark columns).

FIG. 26. Reference neutral N-glycan structures for NMR analysis (A-D).

FIG. 27. Reference acidic N-glycan structures for NMR analysis (A-E).

FIG. 28. Neutral O-glycan fraction glycan signals of cord bloodmononuclear cells (CB MNC).

FIG. 29. Acidic O-glycan fraction glycan signals of cord bloodmononuclear cells (CB MNC).

FIG. 30. Fragmentation mass spectrometry of parent ion at m/z 1765.75corresponding to [M-H+2Na]⁺ adduct ion of Hex5HexNAc4SP1. Fragment ionscorreonding to loss of SPNa (m/z 166322), HexNAcSPNa (m/z 1459.92), orHexHexNAcSPNa (m/z 1298.26) are the major fragmentation products.x-axis: mass-to-charge ratio (m/z); y-axis: relative signal intensity(%).

FIG. 31. FACS analysis of seven cord blood mononuclear cell samples(parallel columns) by FITC-labelled lectins. The percentages refer toproportion of cells binding to lectin. For abbreviations ofFITC-labelled lectins see text.

FIG. 32. Schematic representation of the analysis method of the presentExample. a N-glycans were detached from stem cell glycoproteins byN-glycosidase enzyme digestion. b The total N-glycan pool was purifiedwith microscale solid-phase extraction and divided into neutral andacidic N-glycan fractions. c and d The N-glycan fractions were analyzedby MALDI-TOF mass spectrometry either in positive ion mode as alkalimetal adduct ions (c) or in negative ion mode as deprotonated ions (d).

FIG. 33. Mass spectrometric profiling of human embryonic stem cell anddifferentiated cell N-glycans. a Neutral N-glycans and b 50 mostabundant acidic N-glycans of the four hESC lines (white columns),embryoid bodies derived from FES 29 and FES 30 hESC lines EB, lightcolumns), and stage 3 differentiated cells derived from FES 29 (st3,black columns). The columns indicate the mean abundance of each glycansignal (% of the total detected glycan signals). Error bars indicate therange of detected signal intensities. Proposed monosaccharidecompositions are indicated on the x-axis. H: hexose, N:N-acetylhexosamine, F: deoxyhexose, S: N-acetylneuraminic acid, G:N-glycolylneuraminic acid, P: sulphatelphosphate ester.

FIG. 34. Venn diagram showing distribution of the detected neutral andacidic N-glycan signals a between the four hESC lines (FES) and bbetween hESC, embryoid bodies derived from FES 29 and FES 30 hESC lines(EB), and stage 3 differentiated cells derived from FES 29 (st.3).

FIG. 35. a Classification rules for major human N-glycan biosyntheticgroups. The minimal structures of each biosynthetic group (solid lines)form the basis for the classification rules. Variation of the basicstructures by additional monosaccharide units (dashed lines) genertescomplexity to stem cell glycosylation as revealed in the present study.H: hexose, N: N-acetylhexosamine, F: deoxyhexose, S: N-acetylneurainnicacid. b Pie diagrams showing the classification of human embryonic stemcells (WESC), emblyoid bodies (EB), and stage 3 differentiated cells(st3) data as described in the Examples. c Proportions of the two majoridentified differentiation stage associated glycan features within thecomplex-type sialylated N-glycans according to Table 41.

FIG. 36. Glycan fingerprinting analysis of the four hESC lines, embryoidbodies derived from FES 29 and FES 30 hESC lines (EB), and stage 3differentiated cells derived from FES 29 (st3). The glycan score wascalculated as described in the Examples.

FIG. 37. Latin staining of hESC colonies grown on mouse feeder celllayers, with (A) Maackia amuriensis agglutinin (MAA) that recognizesα2,3-sialylated glycans, and with (B) Pisum sativum agglutinin (PSA)that recognizes α-mannosylated glycans. Lectin binding to hESC wasinhibited by α3′-sialyllactose and D-mannose for MAA and PSA,respectively, and PSA recognized hESC only after cell permeabilization(data not shown). Mouse fibroblasts had complementary staining patternswith both lectins, indicating that their surface glycans differed fromhESC. C. The results indicate that mannosylated N-glycans are localizedin the intracellular compartments in hESC, whereas α2,3-sialylatedglycans occur on the cell surface.

FIG. 38. Implications of hESC fucosyltransferase gene expressionprofile. A. hESC express three fucosyltransferase genes: FUT1, FUT4, andFUT8. B. The expression levels of FUT1 and FUT4 are increased in hESCcompared to EB, which potentially leads to more complex fucosylation inhESC. Known fucosyltransferase glycan products are shown. Arrowsindicate sites of glycan chain elongation. Asn indicates linkage toglycoprotein.

FIG. 39. Portrait of the hESC N-glycome MALDI-TOF mass spectrometricprofiling of the most abundant 50 neutral N-glycans (A.) and 50sialylated N-glycans (B.) of the four bESC lines FES 21, 22, 29, and 30(black columns), four EB samples (gray columns), and four st3differentiated cell samples (white columns) derived from the four hESClines, respectively. The columns indicate the mean abundance of eachglycan signal (% of the total glycan signals). The observed m/z valuesfor either [M+Na]+ or [M-H]− ions for the neutral and sialylatedN-glycan fractions, respectively, are indicated on the x-axis. Proposedmonosaccharide compositions and N-glycan types are presented in Table48.

FIG. 40. Detection of hESC glycans by structure-specific reagents. Tostudy the localization of the detected glycan components in hESC, stemcell colonies grown on mouse feeder cell layers were labeled byfluoresceinated glycan-specific reagents selected based on the analysisresults (FIG. 36). A. The hESC surfaces were stained by Maackiaamurensis agglutinin (MAA), indicating that α2,3-sialylated glycans areabundant on hESC but not on feeder cells (MEF, mouse feeder cells). B.In contrast, the hESC cell surfaces were not stained by Pisum sativumagglutinin (PSA) that recognized mouse feeder cells, indicating thatα-mannosylated glycans are not abundant on hESC surfaces but are presenton mouse feeder cells. C. Addition of 3′-sialyllactose blocks MAAbinding, and D. addition of D-mannose blocks PSA binding.

FIG. 41. hESC-associated glycan signals selected from the 50 mostabundant sialylated N-glycan signals of the analyzed hESC, EB, and st3samples (data taken from FIG. 39.B).

FIG. 42. Differentiated cell associated glycan signals selected from the50 most abundant sialylated N-glycan signals of the analyzed hESC, EB,and st3 samples (data taken from FIG. 39.B)

FIG. 43. Schematic representation of the N-glycan change duringdifferentiation (details do not necessarily refer to actual structures).According to characterization of the Finnish hESC lines FES 21, 22, 29,and 30, hESC differentiation leads to a major change in hESC surfacemolecules. St3 means differentiation stage after EB stage.

FIG. 44. Stern cell nomenclature used to describe the present invention.

FIG. 45. MALDI-TOF mass spectrometric profile of isolated human stemcell neutral glycosphingolipid glycans. x-axis: approximate m/z valuesof [M+Na]⁺ ions as described in Table. y-axis: relative molar abundanceof each glycan component in the profile. hESC, BM MSC, CB MSC, CB MNC:stem cell samples as described in the text.

FIG. 46. MALDI-TOF mass spectometric profile of isolated human stem cellacidic glycosphingolipid glycans. x-axis: approximate m/z values of[M-H]⁻ ions as described in Table. y-axis: relative molar abundance ofeach glycan component in the profile. hESC, BM MSC, CB MSC, CB MNC: stemcell samples as described in the text.

SUMMARY OF THE INVENTION

The present invention is directed to production and analysis of broadglycan mixtures from stem cell samples.

The present invention is specifically directed to glycomes of stem cellsaccording to the invention comprising glycan material withmonosaccharide composition for each of glycan mass components accordingto the Formula I:

R₁Hexβz{R₃}_(n1)HexNAcXyR₂  (1),

wherein X is nothing or a glycosidically linked disaccharide epitopeβ4(Fucα6)_(n)GN, whereinn is 0 or 1;

Hex is Gal or Man or GlcA; HexNAc is GlcNAc or GalNAc;

y is anomeric linkage structure α and/or β or a linkage from aderivatized anomeric carbon,z is linkage position 3 or 4, with the provision that when z is 4, thenHexNAc is GlcNAc and Hex is Man or Hex is Gal or Hex is GlcA, andwhen z is 3, then Hex is GlcA or Gal and HexNAc is GlcNAc or GalNAc;R₁ indicates 1-4 natural type carbohydrate substituents linked to thecore structures,R₂ is reducing end hydroxyl, a chemical reducing end derivative or anatural asparagine linked N-glycoside derivative including asparagines,N-glycoside aminoacids and/or peptides derived from proteins, or anatural serine or threonine linked O-glycoside derivative includingasparagines, N-glycoside aminoacids and/or peptides derived fromproteins;R3 is nothing or a branching structure representing GlcNAcβ6 or anoligosaccharide with GlcNAcβ6 at its reducing end linked to GalNAc, whenHexNAc is GalNAc, or R3 is nothing or Fucα4, when Hex is Gal, HexNAc isGlcNAc, and z is 3, or R3 is nothing or Fucα3, when z is 4.

Typical glycomes comprise of subgroups of glycans, including N-glycans,O-glycans, glycolipid glycans, and neutral and acidic subglycomes.

The preferred analysis method includes:

-   -   1) Preparing a stem cell sample containing glycans for the        analysis    -   2) Releasing total glycans or total glycan groups from a stem        cell sample, or extracting free glycans from a stem cell sample    -   3) Optionally modifying glycans    -   4) Purification of the glycan fraction/fractions from biological        material of the sample    -   5) Optionally modifying glycans    -   6) Analysis of the composition of the released glycans        preferably by mass spectrometry    -   7a) Optionally presenting the data about released glycans        quantitatively and    -   7b) Comparing the quantitative data set with another data set        from another stem cell sample    -   or    -   8) Comparing data about the released glycans quantitatively or        qualitatively with data produced from another stem cell sample

The invention is directed to diagnosis of clinical state of stem cellsamples, based on analysis of glycans present in the samples. Theinvention is especially directed to diagnosing cancer and the clinicalstate of cancer, preferentially to differentiation between stem cellsand cancerous cells and detection of cancerous changes in stem celllines and preparations.

The invention is further directed to structural analysis of glycanmixtures present in stem cell samples.

DESCRIPTION OF THE INVENTION

Glycomes—Novel Glycan Mixtures from Stem Cells

The present invention revealed novel broad mixtures of glycans ofdifferent sizes from stem cells. The stem cells contain glycans rangingfrom small oligosaccharides to large complex structures. The analysisreveals compositions with substantial amounts of numerous components andstructural types. Previously the total glycomes from these rarematerials has not been available and nature of the releasable glycanmixtures, the glycomes, of stem cells has been unknown.

The invention revealed that the glycan structures on cell surfaces varybetween the various populations of the early human cells, the preferredtarget cell populations according to the invention. It was revealed thatthe cell populations contained specifically increased “reporterstructures”.

The glycan structures on cell surfaces in general have been known tohave numerous biological roles. Thus the knowledge about exact glycanmixtures from cell surfaces is important for knowledge about the statusof cells. The invention revealed that multiple conditions (vai changesin conditions or developmental state) affect the cells and cause changesin their glycomes.

Molecular Weight Distribution and Structure Groups of the GlycomesPreferred Monosaccharide Compositions of the Glycomes GeneralCompositions

The inventors were able to release or isolate various glycan fractionsfrom stem cells, which are useful for the characterization of thecellular material. The glycans or major part thereof are releasedpreferably from glycoproteins or glycolipids of human stem cells. Theinvention is specifically directed to such glycan fractions.

The glycan fractions of stem cells comprise typically multiple, at leastabout 10 “glycan mass components” typically corresponding at least tenglycans and in most cases clearly more than 10 glycan structures.

Glycan Mass Components and Corresponding Monosaccharide Compositions

The glycan mass components correspond to certain molecular weightsobservable by mass spectrometry and further correspond to specificmonosaccharide composition or monosaccharide compositions. Eachmonosaccharide component is normally present in a glycan asglycosidically linked monosaccharide residue in the nonreducing end partof glycan and the reducing end monosaccharide may be in free alditolform or modified for example by reduction or conjugated to an reducingend modifying reagent well known in the art or to one, two or severalamino acids in case of glycopeptides. Monosaccharide composition can beobtained from molecular mass in a mass spectrum (glycan mass component)after correcting potential effect of the ion forms observable by thespecific mass spectrometry technologue such asprotonation/deprotonation, Na⁺, K⁺, Li⁺, or other adduct combinations,or isotope pattern derived effects. The monosaccharide compositions arecalculated by fitting mixtures of individual monosaccharide (residue)masses and modification groups to corrected molecular mass of glycanmass component. Typically the molecular mass of fitting composition andthe experimental mass correspond to each other very closely with similarfirst and even second decimals with optimal calibration.

The fitting may be further checked by measuring the experimental massdifference from the smaller and/or larger glycan mass component next inthe putative biosynthetic serie of a glycan type and comparing thedifference with the exact molecular mass of corresponding monosaccharideunit (residue), typically the mass differences of fitting components ina good quality mass spectrum and with correct marking of peaks indecimals, preferaby in second or third decimal of the mass numberdepending on the resolution of the specific mass spectrometric method.For optimal mass accuracy, an internal calibration may be used, wheretwo or more known component's mass peaks are used to re-calculate massesfor each components in the spectrum. Such calibration components arepreferably selected among the most abundant glycan signals present inthe glycan profiles, in the case of human or other animal cell derivedglycan profiles most preferably selected among the most abundant glycansignals present in Figures described in the present invention.

The monosaccharide composition includes monosaccharide component namesand number, typically as subscript, indicating how many of theindividual mass components is present in the monosaccharide composition;and names of assigned modifying groups and numbers indicating theirabundance.

It is further realized that the masses of glycan mass component may beobtained as exact monoisotopic mass of usually smallest isotope of theglycan mass component or as an average mass of the isotope distributionof the glycan mass component Exact mass is calculated form exact massesof individual mass components and average from masses average masses ofindividual mass components. Person skilled in art can recognize from thepeak shapes (i.e. by the resolution obtained) in the mass spectrumwhether to use monoisotopic or average masses to interpret the spectra.It is further realized that average and exact masses can be converted toeach other when isotope abundances of molecules are known, typicallynatural abundance without enrichment of isotopes can be assumed, unlessthe material is deliberately labelled with radioactive or stableisotopes.

It is further realized that specific rounded mass numbers can be used asnames for glycan mass components. The present invention uses preferablymass numbers rounded down from the exact mass of the monosaccharidecomposition (and usually observable or observed mass) to closest integeras names of glycan mass components.

The masses of gylcan mass components are obtained by calculatingmolecular mass of individual monosaccharide components (Hex, HexNAc,dhex, sialic acids) from the known atom compositions (for example hexose(Hex) corresponds to C₆H₁₂O₆) and subtracting for water in case ofmonosaccharide residue, followed by calculating the sum of themonosaccharide components (and possible modifications such as SO₃ orPO₃H) It is further realized that molecular masses of glycans may becalculated from atomic compositions or any other suitable mass unitscorresponding molecular masses of these. The molecular masses andcalculation thereof are known in the art and masses of monosaccharidecomponents/residues are available in tables with multiple decimals fromvarious sources.

It is further realized that many of the individual monosaccharidecompositions described in the present invention further correspond toseveral isomeric individual glycans. In addition, there exist alsomonosaccharide compositions that have nearly equal masses, for exampledHex2 and NeuAc monosaccharide residues that have nearly equal masses,and other examples can be presented by a person skilled in the art. Itis realized that the ability to differentiate compositions with nearlyequal masses depends on instrumentation, and the present method isespecially directed to a possibility to select also such compositions inplace of proposed compositions.

The preferred glycans in glycomes comprise at least two of followingmonosaccharide component residues selected from group: Hexoses (Hex)which are Gal, Glc and Man; N-acetylhexosamines (HexNAc) which areGlcNAc and GalNAc; pentose, which is Xyl; Hexuronic acids which are GlcAand IdoA; deoxyhexoses (dHex), which is fucose and sialic acids whichare NeuAc and/NeuGc; and further modification groups such as acetate(Ac), sulphate and phosphate forming esters with the glycans. Themonosaccharide residues are further grouped as major backbonemonosaccharides including GlcNAc, HaxA, Man and Gal; and specificterminal modifying monosaccharide units Glc, GalNAc, Xyl and sialicacids.

Detection of Glycan Modifications

The present invention is directed to analyzing glycan components frombiological samples, preferably as mass spectrometric signals. Specificglycan modifications can be detected among the detected signals bydetermined indicative signals as exemplified below. Modifications canalso be detected by more specific methods such as chemical or physicalmethods, for example mass spectrometric fragmentation or glycosidasedetection as disclosed in the present invention. In a preferred form ofthe present method, glycan signals are assigned to monosaccharidecompositions based on the detected m/z ratios of the glycan signals, andthe specific glycan modifications can be detected among the detectedmonosaccharide compositions.

In a further aspect of the present invention, relative molar abundancesof glycan components are assigned based on their relative signalintensities detected in mass spectrometry as described in the Examples,which allows for quantification of glycan components with specificmodifications in relation to other glycan components. The present methodis also directed to detecting changes in relative amounts of specificmodifications in cells at different time points to detect changes incell glycan compositions.

Glycome Glycan Fraction Further Comprising Monosaccharides

The invention is specifically directed to glycan compositions, whichfurther comprise at least one monosaccharide component in free form,preferably a preferred monosaccharide component described above. Themonosaccharide comprising compositions are in a preferred embodimentderived from a cell material or released glycomes, which has been incontact with monosaccharide releasing chemicals or enzymes, preferablywith exoglycosidase enzymes or chemicals such as oxidating reagentsand/or acid, more preferably with a glycosidase enzyme. The invention isfurther directed to compositions comprising a specific preferredmonosaccharide according to the invention, an exoglycosidase enzymecapable releasing all or part of the specific monosaccharide and anglycan composition according to the invention from which at least partof the terminal specific monosaccharide has been released.

Limit of Detection for Glycome Components

It is further realized that by increasing the sensitivity of detectionthe number of glycan mass components can be increased. The analysisaccording to the invention can be in most cases performed from major orsignificant components in the glycome mixture. The present invention ispreferably directed to detection of glycan mass components from a highquality glycan preparation with optimised experimental condition, whenthe glycan mass components have abundance at least higher than 0.01% oftotal amount of glycan mass components, more preferably of glycan masscomponents of abundance at least higher than 0.05%, and most preferablyat least higher than 0.10% are detected. The invention is furtherdirected practical quality glycome compositions and analytic processdirected to it, when glycan mass components of at least about 0.5%, oftotal amount of glycan mass components, more preferably of glycan masscomponents of abundance at least higher than 1.0%, even more preferablyat least higher than 2.0%, most preferably at least higher than 4.0%(presenting lower range practical quality glycome), are detected. Theinvention is further directed to glycomes comprising preferred number ofglycan mass components of at least the abundance of observable in highquality glycomes, and in another embodiment glycomes comprisingpreferred number of glycan mass components of at least the abundance ofobservable in practical quality glycomes.

Subglycomes Obtainable by Purification or Specific Release Method

It further realized that fractionation or differential specific releasemethods of glycans from glycoconjugates can be applied to producesubglycomes containing part of glycome.

The subglycomes produced by fractionation of glycomes are called“fractionated subglycomes”.

The glycomes produced by specific release methods are“linkage-subglycomes”. The invention is further directed to combinationsof linkage-subglycomes and fractionated subglycomes to produce“fractionated linkage-subglycomes”, for example preferred fractionatedlinkage-subglycomes includes neutral O-glycans, neutral N-glycans,acidic O-glycans, and acidic N-glycans, which were found very practicalin characterising target material according to the invention.

The fractionation can be used to enrich components of low abundance. Itis realized that enrichment would enhance the detection of rarecomponents. The fractionation methods may be used for larger amounts ofcell material. In a preferred embodiment the glycome is fractionatedbased on the molecular weight, charge or binding to carbohydrate bindingagents.

These methods have been found useful for specific analysis of specificsubglycomes and enrichment more rare components. The present inventionis in a preferred embodiment directed to charge based separation ofneutral and acidic glycans. This method gives for analysis method,preferably mass spectroscopy material of reduced complexity and it isuseful for analysis as neutral molecules in positive mode massspectrometry and negative mode mass spectrometry for acidic glycans.

Differential release methods may be applied to get separately linkagespecific subglycomes such as O-glycan, N-glycan, glycolipid orproteoglycan comprising fractions or combinations thereof. Chemical andenzymatic methods are known for release of specific fractions,furthermore there are methods for simultaneous release of O-glycans andN-glycans.

Novel Complete Compositions

It is realized that at least part of the glycomes have novelty as novelcompositions of very large amount of components. The glycomes comprisingvery broad range substances are referred as complete glycomes.

Preferably the composition is a complete composition comprisingessentially all degrees of polymerisation in general from at least aboutdisaccharides, more preferably from trisaccharides to at least about25-mers in a high resolution case and at least to about 20-mers or atleast about 15-mer in case of medium and practical quality preparations.

It is realized that especially the lower limit, but also upper limit ofa subglycome depend on the type of subglycome and/or method used for itsproduction. Different complete ranges may be produced in scope ofgeneral glycomes by fractionation, especially based on size of themolecules.

Novel Compositions with New Combinations of Subglycomes and PreferredGlycan Groups

It is realized that several glycan types are present as novel glycomecompositions produced from the stem cells. The invention is specificallydirected to novel mixture composition comprising different subglycomesand preferred glycan groups

Novel Quantitative Glycome Compositions

It is realised that the glycome compossitiona as descried in examplesrepresent quantitatively new data about glycomes from the preferred stemcell types. The proportions of various components cannot be derived frombackground data and are very useful for the analysis methods accordingto the invention. The invention is specifically directed to glycomecompositions according to the examples when the glycan mass componentsare present in essentially similar relative amounts.

Preferred Composition Formulas

The present invention is specifically directed to glycomes of stem cellsaccording to the invention comprising glycan material withmonosaccharide composition for each of glycan mass components accordingto the Formula I:

NeuAc_(m)NeuGc_(n)Hex_(o)HexNAc_(p)dHex_(q)HexA_(r)Pen_(s)Ac_(t)ModX_(x)  (I)

where m, n, o, p, q, r, s, t, and x are independent integers with values≧0 and less than about 100,with the proviso thatfor each glycan mass components at least two of the backbonemonosaccharide variables o, p, or r is greater than 0, andModX represents a modification (or N different modifications Mod1, Mod2,. . . , ModN), present in the composition in an amount of x (or inindependent amounts of x1, x2, . . . , xN), Preferably examples of suchmodifications (Mod) including for example SO₃ or PO₃H indicating estersof sulfate and phosphate, respectivelyand the glycan composition is preferably derived from isolated humanstem cells or preferred subpopulations thereof according to theinvention.

It is realized that usually glycomes contain glycan material for whichthe variables are less much less than 100, but large figures may beobtained for polymeric material comprising glycomes with repeatingpolymer structures, for example ones comprising glycosaminoglycan typematerials. It is further realized that abundance of the glycan masscomponents with variables more than 10 or 15 is in general very low andobservation of the glycome components may require purification andenrichment of larger glycome components from large amounts of samples.

Broad Mass Range Glycomes

In a preferred embodiment the invention is directed to broad mass rangeglycomes comprising polymeric materials and rare individual componentsas indicated above. Observation of large molecular weight components mayrequire enrichment of large molecular weight molecules comprisingfraction. The broad general compositions according to the Formula I areas described above,

with the proviso thatm, n, o, p, q, r, s, t, and x are independent integers with preferablevalues between 0 and 50, with the proviso that for each glycan masscomponents at least two of o, p, or r is at least 1, and the sum of themonosaccharide variables; m, n, o, p, q, r, and s, indicating the degreeof polymerization or oligomerization, for each glycan mass component isless than about 100 and the glycome comprises at least about 20different glycans of at least disaccharides.

Practical Mass Range Glycomes

In a preferred embodiment the invention is directed to practical massrange and high quality glycomes comprising lower molecular weight rangesof polymeric material. The lower molecular weight materials at least inpart and for preferred uses are observable by mass spectrometry withoutenrichment.

In a more preferred general composition according to the Formula I asdescribed above, m, n, o, p, q, r, s, t, and x are independent integerswith preferable values between 0 and about 20, more preferably between 0and about 15, even more preferably between 0 and about 10, with theproviso that at least two of o, p, or r is at least 1,

and the sum of the monosaccharide variables; m, n, o, p, q, r, and s,indicating the degree of polymerization or oligomerization, for eachglycan mass component is less than about 50 and more preferably lessthan about 30,and the glycome comprises at least about 50 different glycans of atleast trisaccharides.

In a preferred embodiment the invention is directed to practical massrange high quality glycomes which may comprise some lower molecularweight ranges of polymeric material. The lower molecular weightmaterials at least in part and for preferred uses are observable by massspectrometry without enrichment.

In a more preferred general composition according to the Formula I asdescribed above, m, n, o, p, q, r, s, t, and x are independent integerswith preferable values between 0 and about 10, more preferably between 0and about 9, even more preferably, between 0 and about 8, with theproviso that at least two of o, p, or r is at least 1,

and the sum of the monosaccharide variables; m, n, o, p, q, r, and s,indicating the degree of polymerization or oligomerization, for eachglycan mass component is less than about 30 and more preferably lessthan about 25,and the glycome comprises at least about 50 different glycans of atleast trisaccharides.

The practical mass range glycomes may typically comprise tens ofcomponents, for example in positive ion mode MALDI-TOF mass spectrometryfor neutral subglycomes it is usually possible to observe even more than50 molecular mass components, even more than 100 mass componentcorresponding to much larger number of potentially isomeric glycans. Thenumber of components detected depends on sample size and detectionmethod.

Preferred Subglycomes

The present invention is specifically directed to subglycomes of stemcell glycomes according to the invention comprising glycan material withmonosaccharide compositions for each of glycan mass components accordingto the Formula I and as defined for broad and practical mass rangeglycomes. Each subglycome has additional characteristics based on glycancore structures of linkage-glycomes or fractionation method used for thefractionated glycomes. The preferred linkage glycomes includes:

N-glycans, O-glycans, glycolipid glycans, neutral and acidicsubglycomes,

N-glycan Subglycome

Protein N-glycosidase releases N-glycans comprising typically twoN-acetylglycosamine units in the core, optionally a core linked fucoseunit and typically then 2-3 hexoses (core mannoses), after which thestructures may further comprise hexoses being mannose or in complex-typeN-glycans further N-acetylglycosamines and optionally hexoses and sialicacids.

N-glycan subglycomes relased by protein N-glycosidase comprise N-glycanscontaining N-glycan core structure and are releasable by proteinN-glycosidase from cells.

The N-glycan core structure is Manβ4GlcNAcβ(Fucα6)_(n)4GlcNAc, wherein nis 0 or 1 and the N-glycan structures can be elongated from the Manβ4with additional mannosylresidues. The protein N-glycosidase cleaves thereducing end GlcNAc from Asn in proteins. N-glycan subglycomes releasedby endo-type N-glycosidases cleaving between GlcNAc units containManβ4GlcNAcβ-core, and the N-glycan structures can be elongated from theManβ4 with additional mannosylresidues.

In case the Subglycome and analysis representing it as Glycan profile isformed from N-glycans liberated by N-glycosidase enzyme, the preferredadditional constraints for Formula I are:

p>0, more preferably 1≦p≦100, typically p is between 2 and about 20, butpolymeric structures containing glycomes may comprise larger amounts ofHexNAc and it is relaised that in typical core of N-glycans indicatingpresence of at least partially complex type structurewhen p≧3 it follows that o≧1.

Glycolipid Subglycome

In case the Subglycome and analysis representing it as Glycan profile isformed from lipid-linked glycans liberated by endoglycoceramidaseenzyme, the preferred additional constraints for Formula I are:

o>0, more preferably 1≦o≦100, andwhen p≧1 it follows that o≧2.

Typically glycolipids comprise two hexoses (a lactosylresidue) at thecore. The degree of oligomerization in a usual practical glycome fromglycolipds is under about 20 and more preferably under 10. Very largestructures comprising glycolipids, polyglycosylceramides, may needenrichment for effective detection.

Neutral and Acidic Subglycomes

Most preferred fractionated Subglycomes includes 1) subglycome ofneutral glycans and 2) subglycome of acidic glycans. The major acidicmonosaccharide unit is in most cases a sialic acid, the acidic fractionrnay further comprise natural negatively charged structure/structuressuch as sulphate(s) and/phosphate(s).

In case the Subglycome and analysis representing it as Glycan profile isformed from sialylated glycans, the preferred additional constraints forFormula I are: (m+n)>0, more preferably 1≦(m+n)≦100.

Large amounts of sialic acid in a glycan mass component would indicatepresence of polysailic acid type structures. Practical and highresolutions acidic glycomes usually have m+n values for individual majorglycan mass components with preferred abundance between 1 and 10, morepreferably and of the between 1-5 and most preferably between 14 for ausual glycomes according to the invention. For neutral glycans, (m+n)=0,and they do not contain negatively charged groups as above.

Preferred Structure Groups Observable in Glycome Profiles

The present invention is specifically directed to the glycomes of stemcell according to the invention comprising as major components at leastone of structure groups selected from the groups described below.

Glycan Groups

According to the present invention, the Glycan signals are optionallyorganized into Glycan groups and Glycan group profiles based on analysisand classification of the assigned monosaccharide and modificationcompositions and the relative amounts of monosaccharide and modificationunits in the compositions, according to the following classificationrules:

-   -   1° The glycan structures are described by the formulae:

Hex_(m)HexNAc_(n)dHex_(o)NeuAc_(p)NeuGc_(q)Pen_(r)Mod1_(sMod1)Mod2_(sMod2). . . ModX_(sModX),

-   -   -   wherein m, n, o, p, q, individual sMod, and X are each            independent variables, and Mod is a functional group            covalently linked to the glycan structure.

    -   2° Glycan structures in general are classified as follows:

    -   a. Structures (p,q=0) are classified as “non-sialylated”,

    -   b. Structures (p,q>0) are classified as “sialylated”,

    -   c. Structures (q>0) are classified as “NeuGc-containing”,

    -   d. Relation [2 (p+q): (m+n)] describes the general sialylation        degree of a glycan structure,

    -   e. In the case of mrnammalian glycans, structures (o=0) are        classified as “non-fucosylated”,

    -   f. In the case of mammalian glycans, structures (o>0) are        classified as “fucosylated”,

    -   g. Structures (Mod=Ac and sAc>0) are classified as ‘acetylated’,

    -   h. Structures (Mod=SO₃ and sSO₃>0) are classified as ‘sulfated’,        and

    -   i. Structures (Mod=PO₃H and sPO₃H>0) are classified as        ‘phosphorylated’.

    -   3° N-glycan glycan structures, generated e.g. by the action of        peptide-N-glycosidases, are classified as follows:

    -   a. Structures (n=2 and m>0 and p,q=0) are classified as        “mannose-terminated N-glycans”,

    -   b. Structures (n=2 and m>5 and o,p,q=0) are classified as        “high-mannose N-glycans”,

    -   c. Structures (n=2 and m>5 and o>0 and p,q=0) are classified as        “fucosylated high-mannose N-glycans”,

    -   d. Structures (n=2 and 4≧m≧1 and p,q=0) are classified as        “low-mannose N-glycans”,

    -   e. Structures (n=2 and 4≧m≧1 and o>0 and p,q=0) are classified        as “fucosylated low-mannose N-glycans”,

    -   f. Structures (n=3 and m≧2) are classified as “hybrid-type or        monoantennary N-glycans”,

    -   g. Structures (n≧4 and m≧3) are classified as “complex-type        N-glycans”,

    -   h. Structures (n>m≧2) are classified as “N-glycans containing        non-reducing terminal N-acetylhexosamine”,

    -   i. Structures (n=m≧5) are classified as “N-glycans potentially        containing bisecting N-acetylglucosamine”,

    -   j. In the case of mammalian N-glycans, structures (o≧2) are        classified as “N-glycans containing α2-, α3-, or α4-linked        fucose”,

    -   k. Relation [2 (p+q): (m+n−5)] describes the “overall        sialylation degree” of a sialylated N-glycan structure, and

    -   l. Specifically, sum (p+q) describes the “sialylation degree” of        a sialylated hybrid-type or monoantennary N-glycan structure.

    -   4′ Mucin-type O-glycan structures, generated e.g. by alkaline        β-elimination, are classified as follows:        -   a. Structures (n=m), with (N=n=m), are classified as “Type N            O-glycans”,        -   b. More specifically, structures (n=m=1) are classified as            “Type 1 O-glycans”,        -   c. More specifically, structures (n=m=2) are classified as            “Type 2 O-glycans”,        -   d. More specifically, structures (n=m=3) are classified as            “Type 3 O-glycans”,        -   e. Relation [2 (p+q): (m+n)] describes the overall            sialylation degree of a sialylated N-glycan structure, and        -   f. Specifically, relation [(p+q): N] describes the            sialylation degree of a sialylated Type N O-glycan            structure.

Lipid-linked can also be classified into structural groups based ontheir monosaccharide compositions, as adopted from the classificationsabove according to the invention.

-   -   For example, glycan signal corresponding to a human stem cell        N-glycan structure:        -   Hex₅HexNAc₄dHex₂NeuAc₁Ac₁,    -   is classified as belonging to the following Glycan Groups:        -   sialylated (general sialylation degree: 2/9),        -   fucosylated,        -   acetylated,        -   complex-type N-glycans (overall sialylation degree: 0.5),        -   N-glycans containing α2-, α3-, or α4-linked fucose.

Glycomes Comprising Novel Glycan Types

The present invention revealed novel unexpected components among in theglycomes studied. The present invention is especially directed toglycomes comprising such unusual materials

Preferred Glycome Types Derivatized Glycomes

It is further realized that the glycans may be derivatized chemicallyduring the process of release and isolation. Preferred modificationsinclude modifications of the reducing end and or modifications directedespecially to the hydroxyls- and/or N-atoms of the molecules. Thereducing end modifications include modifications of reducing end ofglycans involving known derivatization reactions, preferably reduction,glycosylamine, glycosylamide, oxime (aminooxy-) and reductive aminationmodifications. Most preferred modifications include modification of thereducing end. The derivatization of hydroxyl- and/or amine groups, suchas produced by methylation or acetylation methods includingpermethylation and peracetylation has been found especially detrimentalto the quantitative relation between natural glycome and the releasedglycome.

Non-derivatized Released Glycomes

In a preferred embodiment the invention is directed to non-derivatizedreleased glycomes. The benefit of the non-derivatized glycomes is thatless processing needed for the production. The non-derivatized releasedglycomes correspond more exactly to the natural glycomes from whichthese are released. The present invention is further directed toquantitative purification according to the invention for thenon-derivatized releases glycomes and analysis thereof.

The present invention is especially directed to released glycomes whenthe released glycome is not a permodified glycorne such as permethylatedglycome or peracetyated glycome. The released glycome is more preferablyreducing end derivatized glycome or a non derivatized glycome, mostpreferably non-derivatized glycome.

Novel Cell Surface Glycomes and Released Glycomes of the Target Material

The present invention is further directed to novel total compositions ofglycans or oligosaccharides referred as glycomes and in a more specificembodiment as released glycomes observed from or produced from thetarget material according to the invention. The released glycomeindicates the total released glycans or total specific glycansubfractions released from the target material according to theinvention. The present invention is specifically directed to releasedglycomes meaning glycans released from the target material according tothe invention and to the methods according to the invention directed tothe glycomes.

The present invention preferably directed to the glycomes released astruncated and/or non-truncated glycans and/or derivatized according tothe invention.

The invention is especially directed to N-linked and/or O-linked and/orLipid linked released glycomes from the target material according to theinvention. The invention is more preferably directed to releasedglycomes comprising glycan structures according to the invention,preferably glycan structures as defined in formula I. The invention ismore preferably directed to N-linked released glycomes comprising glycanstructures according to the invention, preferably glycan structures asdefined in formula I.

Non-derivatized Released Cell Surface Glycomes and Production

In a preferred embodiment the invention is directed to non-derivatizedreleased cell surface glycomes. The non-derivatized released cellsurface glycomes correspond more exactly to the fractions of glycomesthat are localized on the cell surfaces, and thus available forbiological interactions. These cell surface localized glycans are ofespecial importance due to their availability for biologicalinteractions as well as targets for reagents (e.g. antibodies, lectinsetc. . . . ) targeting the cells or tissues of interest. The inventionis further directed to release of the cell surface glycomes, preferablyfrom intact cells by hydrolytic enzymes such as proteolytic enzymes,including proteinases and proteases, and/or glycan releasing enzymes,including endo-glycosidases or protein N-glycosidases. Preferably thesurface glycoproteins are cleaved by proteinase such as trypsin and thenglycans are analysed as glycopeptides or preferably relased further byglycan relasing enzyme.

Analysis of the Glycomes

Analysis of the glycan mixtures by physical means, preferably by massspectrometry The present invention is directed to analysis of glycanmixtures present in stem cell samples.

Quantitative and Qualitative Analysis of Glycan Profile Data

The invention is directed to novel methods for qualitative analysis ofglycome data. The inventors noticed that there are specific componentsin glycomes according to the invention, the presence or absence of whichare connected or associated with specific cell type or cell status. Itis realized that qualitative comparison about the presence of absence ofsuch signals are useful for glycome analysis. It is further realizedthat signals either present or absent that are derived from a generalglycome analysis may be selected to more directed assay measuring onlythe qualitatively changing component or components optionally with amore common component or components useful for verification of dataabout the presence or absence of the qualitative signal.

The present invention is further specifically directed to quantitativeanalysis of glycan data from stem cell samples. The inventors noted thatquantitative comparisons of the relative abundances of the glycomecomponents reveal substantial differences about the glycomes useful forthe analysis according to the invention.

Essential Steps of the Glycome Analysis

The process contains essential key steps which should be included inevery process according to the present invention.

The essential key steps of the analysis are:

-   -   1. Release of total glycans or total glycan groups from a stem        cell sample    -   2. Purification of the glycan fraction/fractions from biological        material of the sample, preferably by a small scale column array        or an array of solid-phase extraction steps    -   3. Analysis of the composition of the released glycans,        preferably by mass spectrometry

In most cases it is useful to compare the data with control sample data.The control sample may be for example from a healthy tissue or cell typeand the sample from same tissue altered by cancer or another disease. Itis preferable to compare samples from same individual organism,preferably from the same human individual.

Specific Types of the Glycome Analysis Comparative Analysis

The steps of a comparative analysis are:

-   -   1. Release of total glycans or total glycan groups from a cell        sample    -   2. Purification of the glycan fraction/fractions from biological        material of the sample, preferably by a small scale column array        or an array of solid-phase extraction steps    -   3. Analysis of the composition of the released glycans,        preferably by mass spectrometry    -   4. Comparing data about the released glycans quantitatively or        qualitatively with data produced from another cell sample

It may be useful to analyse the glycan structural motifs present in thesample, as well as their relative abundances. The ability to elucidatestructural motifs results from the quantitative nature of the presentanalysis procedure, comparison of the data to data from previouslyanalyzed samples, and knowledge of glycan biosynthesis.

Analysis Including Characterization of Structural Motives

The glycome analysis may include characterization of structural motivesof released glycans. The structural motif analysis may be performed incombination with structural analysis.

Preferred methods to reveal specific structural motifs include

-   -   a) direct analysis of specific structural modifications of the        treatment of glycans preferably by exo- or endoglycosidases        and/or chemical modification or    -   b) indirect analysis by analysis of correlating factors for the        structural motives for such as mRNA-expression levels of        glycosyltransferases or enzymes producing sugar donor molecules        for glycosyltransferases.

The direct analyses are preferred as they are in general more effectiveand usually more quantitative methods, which can be combined to glycomeanalysis.

In a preferred embodiment the invention is directed to combination ofanalysis of structural motifs and glycome analysis.

The steps of a structural motif analysis are:

-   -   1. Release of total glycans or total glycan groups from a stem        cell sample    -   2. Purification of the glycan fraction/fractions from biological        material of the sample, preferably by a small scale column array        or an array of solid-phase extraction steps    -   3. Analysis of the composition of the released glycans,        preferably by mass spectrometry    -   4. Analysis of structural motifs present in of the glycan        mixture, and optionally their relative abundancies    -   5. Optionally, comparing data about the glycan structural motifs        with data produced from another stem cell sample

The steps 3 and 4 may be combined or performed in order first 4 and then3.

Preferred Detailed Glycome Analysis Including Quantative Data Analysis

Detailed preferred glycome analysis according to the invention

More detailed preferred analysis method include following analysissteps:

-   -   1. Preparing a stem cell sample containing glycans for the        analysis    -   2. Release total glycans or total glycan groups from a stem cell        sample    -   3. Optionally modifying glycans or part of the glycans.    -   4. Purification of the glycan fraction/fractions from biological        material and reagents of the sample by a small scale column        array    -   5. Optionally modifying glycans and optionally purifying        modified glycans    -   6. Analysis of the composition of the released glycans        preferably by mass spectrometry using at least one mass        spectrometric analysis method    -   7. a) Optionally presenting the data about released glycans        quantitatively and    -   7. b) Comparing the quantitative data set with another data set        from another stem cell sample    -   and/or alternatively to 7a) and 7b)    -   8. Comparing data about the released glycans quantitatively or        qualitatively with data produced from another stem cell sample

The present methods further allow the possibility to use part of thenon-modified material or material modified in step 3 or 5 for additionalmodification step or step and optionally purified after modificationstep or steps, optionally combining modified samples, and analysis ofadditionally modified samples, and comparing results from differentiallymodified samples.

As mentioned above, It is realized that many of the individualmonosaccharide compositions in a given glycome further corresponds toseveral isomeric individual glycans. The present methods allow forgeneration of modified glycomes. This is of particular use whenmodifications are used to reveal such information about glycomes ofinterest that is not directly available from a glycan profile alone (orglycome profiles to compare). Modifications can include selectiveremoval of particular monosaccharides bound to the glycome by a definedglycosidic bond, by degradation by specific exoglycosidases or selectivechemical degradation steps such as e.g. periodic acid oxidation.Modifications can also be introduced by using selectiveglycosyltransferase reactions to label the free acceptor structures inglycomes and thereby introduction of a specific mass label to suchstructures that can act as acceptors for the given enzyme. In preferredembodiment several of such modifications steps are combined and used toglycomes to be compared to gain further insights of glycomes and tofacilitate their comparison.

Quantitative Presentation of Glycome Analysis

The present invention is specifically directed to quantitativepresentation of glycome data.

Two-dimensional Presentation by Quantitation and Component Indicators

The quantitative presentation means presenting quantitative signals ofcomponents of the glycome, preferably all major components of theglycome, as a two-dimensional presentation including preferably a singlequantitative indicator presented together with component identifier.

The preferred two dimensional presentations includes tables and graphspresenting the two dimensional data The preferred tables listquantitative indicators in connection with, preferably beside or underor above the component identifiers, most preferably beside theidentifier because in this format the data comprising usually largenumber of component identifier—quantitation indicator pairs.

Quantitation Indicator

The quantitation indicator is a value indicating the relative abundanceof the single glycome component with regard to other components of totalglycome or subglycome. The quantitation indicator can be directlyderived from qualitative experimental data, or experimental datacorrected to be quantitative.

Normalized Quantitation Indicator

The quantitation indicator is preferably a normalized quantitationindicator. The normalized quantitation indicator is defined as theexperimental value of a single experimental quantitation indicatordivided by total sum of quantitation indicators multiplied by a constantquantitation factor.

Preferred quantitation factors include integer numbers from 1-1000 0000000, more preferably integer numbers 1, 10 or 100, and more preferably 1or 100, most preferably 100. The quantitation number one is preferred ascommonly understandable portion from 1 concept and the most preferredquantitation factor 100 corresponds to common concept of percent values.

The quantitation indicators in tables are preferably rounded tocorrespond to practical accuracy of the measurements from which thevalues are derived from. Preferred rounding includes 2-5 meaningfulaccuracy numbers, more preferably 24 numbers and most preferably 2-3numbers.

Component Indicators

The preferred component indicators may be experimentally derivedcomponent indicators. Preferred components indicators in the context ofmass spectrometric analysis includes mass numbers of the glycomecomponents, monosaccharide or other chemical compositions of thecomponents and abbreviation corresponding to thereof names of themolecules preferably selected from the group: desriptive names andabbreviations; chemical names, abbreviations and codes; and molecularformulas including gaphic representations of the formulas. It is furtherrealized that molecular mass based component indicators may includemultiple isomeric structures. The invention is in a preferred embodimentdirected to practical analysis using molecular mass based componentindicators. In more specific embodiment the invention is furtherdirected to chemical or enzymatic modification methods or indirectmethods according to the invention in order to resolve all or part ofthe isomeric components corresponding to a molecular mass basedcomponent indicators.

Glycan Signals

The present invention is directed to a method of accurately defining themolecular masses of glycans present in a sample, and assigningmonosaccharide compositions to the detected glycan signals.

The Glycan signals according to the present invention are glycancomponents characterized by:

1° mass-to-charge ratio (m/z) of the detected glycan ion,2° molecular mass of the detected glycan component, and/or3° monosaccharide composition proposed for the glycan component

Glycan Profiles

The present invention is further directed to a method of describing massspectrometric raw data of Glycan signals as two-dimensional tables of:

1° monosaccharide composition, and2° relative abundance,which form the Glycan profiles according to the invention.Monosaccharide compositions are as described above. For obtainingrelative abundance values for each Glycan signal, the raw data isrecorded in such manner that the relative signal intensities of theglycan signals represent their relative molar proportions in the sample.Methods for relative quantitation in MALDI-TOF mass spectrometry ofglycans are known in the art (Naven & Harvey, 19xx; Papac et al., 1996)and are described in the present invention. However, the relative signalintensities of each Glycan signal are preferably corrected by talinginto account the potential artefacts caused by e.g. isotopicoverlapping, alkali metal adduct overlapping, and other disturbances inthe raw data, as described below.

By forming these Glycan profiles and using them instead of the raw data,analysis of the biological data carried by the Glycan profiles isimproved, including for example the following operations:

1° identification of glycan signals present in the glycan profile,2° comparison of glycan profiles obtained from different samples,3° comparison of relative intensities of glycan signals within theglycan profile, and4° organizing the glycan signals present in the glycan profile intosubgroups or subprofiles.

Analysis of Associated Signals to Produce Single Quantitative Signal(Quantitation Indicator) Analysis of Associated Signals: IsotopeCorrection

Glycan signals and their associated signals may have overlapping isotopepatterns.

Overlapping of isotope patterns is corrected by calculating theexperimental isotope patterns and subtracting overlapping isotopesignals from the processed data.

Analysis of Associated Signals: Adduct Ion Correction in Positive IonMode

Glycan signals may be associated with signals arising from multipleadduct ions in positive ion mode, e.g. different alkali metal adductions. Different Glycan signals may give rise to adduct ions with similarm/z ratios: as an example, the adduct ions [Hex+Na]⁺ and [dHex+K]⁺ havem/z ratios of 203.05 and 203.03, respectively. Overlapping of adductions is corrected by calculating the experimental alkali metal adduction ratios in the sample and using them to correct the relativeintensities of those Glycan signals that have overlapping adduct ions inthe experimental data Preferably, the major adduct ion type is used forcomparison of relative signal intensities of the Glycan signals, and theminor adduct ion types are removed from the processed data. Thecalculated proportions of minor adduct ion types are subtracted from theprocessed data

Analysis of Associated Signals: Adduct Ion Correction in Negative IonMode

Also in negative ion mode mass spectrometry, Glycan signals may beassociated with signals arising from multiple adduct ions. Typically,this occurs with Glycan signals that correspond to multiple acidic groupcontaining glycan structures. As an example, the adduct ions[NeuAc₂—H+Na]⁻ at m/z 621.2 and [NeuAc₂-H+ K]⁻ at m/z 637.1, areassociated with the Glycan signal [NeuAc₂-H]⁻ at m/z 599.2. These adduction signals are added to the Glycan signal and thereafter removed fromthe processed data In cases where different Glycan signals and adduction signals overlap, this is corrected by calculating the experimentalalkali metal adduct ion ratios in the sample and using them to correctthe relative intensities of those Glycan signals that have overlappingadduct ions in the experimental data.

Analysis of Associated Signals: Removal of Elimination Products

Glycan signals may be associated with signals, e.g. elimination of water(loss of H₂O), or lack of methyl ether or ester groups (effective lossof CH₂), resulting in experimental m/z values 18 or 14 mass unitssmaller than the Glycan signal, respectively. These signals are nottreated as individual Glycan signals, but are instead treated asassociated signals and removed from the processed data.

Classification of Glycan Signals into Glycan Groups

According to the present invention, the Glycan signals are optionallyorganized into Glycan groups and Glycan group profiles based on analysisand classification of the assigned monosaccharide and modificationcompositions and the relative amounts of monosaccharide and modificationunits in the compositions, according to the classification rulesdescribed above:

Generation of Glycan Group Profiles.

To generate Glycan group profiles, the proportions of individual Glycansignals belonging to each Glycan group are summed. The proportion ofeach Glycan group of the total Glycan signals equals its prevalence inthe Glycan profile. The Glycan group profiles of two or more samples canbe compared. The Glycan group profiles can be further analyzed byarranging Glycan groups into subprofiles, and analyzing the relativeproportions of different Glycan groups in the subprofiles. Similarlyformed subprofiles of two or more samples can be compared.

Specific Technical Aspects of Stem Cell Glycome Analysis PreferredSample Sizes

The present invention is especially useful when low sample amounts areavailable. Practical cellular or tissue material may be available forexample for diagnostic only in very small amounts.

Sample Sizes for Preferred Pico-scale Preparation Methods

The inventors found surprisingly that glycan fraction could be producedand analysed effectively from samples containing low amount of material,for example 100 000-1 000 000 cells or a cubic millimetre (microliter)of the cells.

The combination of very challenging biological samples and very lowamounts of samples forms another challenge for the present analyticmethod. The yield of the purification process must be very high. Theestimated yields of the glycan fraction of the analytical processesaccording to the present invention varies between about 50% and 99%.Combined with effective removal of the contaminating various biologicalmaterials even more effectively over the wide preferred mass rangesaccording to the present invention show the ultimate performance of themethod according to the present invention.

Isolation of Glycans and Glycan Fractions

The present invention is directed to a method of preparing anessentially unmodified glycan sample for analysis from the glycanspresent in a given sample.

A preferred glycan preparation process consists of the following steps:

1° isolating a glycan-containing fraction from the sample,2° . . . Optionally purification the fraction to useful purity forglycome analysis

The preferred isolation method is chosen according to the desired glycanfraction to be analyzed. The isolation method may be either one or acombination of the following methods, or other fractionation methodsthat yield fractions of the original sample:

1° extraction with water or other hydrophilic solvent, yieldingwater-soluble glycans or glycoconjugates such as free oligosaccharidesor glycopeptides,2° extraction with hydrophobic solvent, yielding hydrophilicglycoconjugates such as glycolipids,3° N-glycosidase treatment, especially Flavobacterium meningosepticumN-glycosidase F treatment, yielding N-glycans,4° alkaline treatment, such as mild (e.g. 0.1 M) sodium hydroxide orconcentrated ammonia treatment, either with or without a reductive agentsuch as borohydride, in the former case in the presence of a protectingagent such as carbonate, yielding β-elimination products such asO-glycans and/or other elimination products such as N-glycans,5° endoglycosidase treatment, such as endo-α-galactosidase treatment,especially Escherichia freundii endo-β-galactosidase treatment, yieldingfragments from poly-N-acetyllactosamine glycan chains, or similarproducts according to the enzyme specificity, and/or6° protease treatment, such as broad-range or specific proteasetreatment, especially trypsin treatment, yielding proteolytic fragmentssuch as glycopeptides.

The released glycans are optionally divided into sialylated andnon-sialylated subfractions and analyzed separately. According to thepresent invention, this is preferred for improved detection of neutralglycan components, especially when they are rare in the sample to beanalyzed, and/or the amount or quality of the sample is low. Preferably,this glycan fractionation is accomplished by graphite chromatography.

According to the present invention, sialylated glycans are optionallymodified in such manner that they are isolated together with thenon-sialylated glycan fraction in the non-sialylated glycan specificisolation procedure described above, resulting in improved detectionsimultaneously to both non-sialylated and sialylated glycan components.Preferably, the modification is done before the non-sialylated glycanspecific isolation procedure. Preferred modification processes includeneuraminidase treatment and derivatization of the sialic acid carboxylgroup, while preferred derivatization processes include amidation andesterification of the carboxyl group.

Glycan Release Methods

The preferred glycan release methods include, but are not limited to,the following methods:

Free glycans—extraction of free glycans with for example water orsuitable water-solvent mixtures.Protein-linked glycans including O- and N-linked glycans—alkalineelimination of protein-linked glycans, optionally with subsequentreduction of the liberated glycans.Mucin-type and other Ser/Thr O-linked glycans—alkaline β-elimination ofglycans, optionally with subsequent reduction of the liberated glycans.N-glycans—enzymatic liberation, optionally with N-glycosidase enzymesincluding for example N-glycosidase F from C. meningosepticum,Endoglycosidase H from Streptomyces, or N-glycosidase A from almonds.Lipid-linked glycans including glycosphingolipids—enzymatic liberationwith endoglycoceramidase enzyme; chemical liberation; ozonolyticliberation.Glycosaminoglycans—treatment with endo-glycosidase cleavingglycosaminoglycans such as chondroinases, chondroitin lyases,hyalurondases, heparanases, heparatinases, orkeratanases/endo-beta-galactosidases; or use of O-glycan release methodsfor O-glycosidic Glycosaminoglycans; or N-glycan release methods forN-glycosidic glycosaminoglycans or use of enzymes cleaving specificglycosaminoglycan core structures; or specific chemical nitrous acidcleavage methods especially for amine/N-sulphate comprisingglycosaminoglycansGlycan fragments—specific exo- or endoglycosidase enzymes including forexample keratanase, endo-β-galactosidase, hyaluronidase, sialidase, orother exo- and endoglycosidase enzyme; chemical cleavage methods;physical methods

Effective Purification Process

The invention describes special purification methods for glycan mixturesfrom tissue samples. Previous glycan sample purification methods haverequired large amounts of material and involved often numerouschromatographic steps and even purification of specific proteins. It isknown that protein glycosylation varies protein specifically and singleprotein specific data can thus not indicate the total tissue levelglycosylation. Purification of single protein is a totally differenttask than purifying the glycan fraction according to the presentinvention.

When the purification starts from a tissue or cells, the old processesof prior art involve often laborious homogenisation steps affecting thequality of the material produced. The present purification directly froma biological sample such as cell or tissue material, involves only a fewsteps and allows quick purification directly from the biologicalmaterial to analysis preferably by mass spectrometry.

Purification from Cellular Materials of Cells and/or Tissues

The cellular material contains various membranes, small metabolites,various ionic materials, lipids, peptides, proteins etc. All of thematerials can prevent glycan analysis by mass spectrometry if thesecannot be separated from the glycan fraction. Moreover, for examplepeptide or lipid materials may give rise to mass spectrometric signalswithin the preferred mass range within which glycans are analysed. Manymass spectrometric methods, including preferred MALDI-mass spectrometryfor free glycan fractions, are more sensitive for peptides than glycans.With the MALDI method peptides in the sample may be analysed withapproximately 1000-fold higher sensitivity in comparision to methods forglycans. Therefore the method according to the present invention shouldbe able to remove for example potential peptide contaminations from freeglycan fractions most effectively. The method should remove essentialpeptide contaminations from the whole preferred mass range to beanalysed.

Purification Suitable for Mass Spectrometry, Especially MALDI-TOF MassSpectometry

The inventors discovered that the simple purification methods wouldseparate released glycans from all possible cell materials so that

1) The sample is technically suitable for mass spectrometric analysis.

-   -   This includes two major properties,    -   a) the samples is soluble for preparation of mass spectrometry        sample and    -   b) does not have negative interactions with chemicals involved        in the mass spectrometric method, preferably the sample dries or        crystallizes properly with matrix chemical used in MALDI-TOF        mass spectrometry

When using MALDI-technologies, the sample does not dry or crystallizeproperly if the sample contains harmful impurity material in asignificant amount

2) The purity allows production of mass spectrum of suitable quality.

-   -   a) The sample has so low level of impurities that it gives mass        spectrometric signals. Especially when using MALDI-TOF mass        spectrometry, signals can be suppressed by background so that        multiple components/peaks cannot be obtained.    -   b) the sample is purified so that there is no major impurity        signals in the preferred mass ranges to be measured.

Preferably the present invention is directed to analysis of unusuallysmall sample amounts. This provides a clear benefit over prior art, whenthere is small amount amount of sample available from a small region ofdiseased tissue or diagnostic sample such as tissue slice produced formicroscopy or biopsy sample. Methods to achieve such purity (puritybeing a requirement for the sensitivity needed for such small sampleamounts) from tissue or cell samples (or any other complex biologicalmatices e.g. serum, saliva) has not been described in the prior art.

In a preferred embodiment the method includes use of non-derived glycansand avoiding general derived glycans. There are methods of producingglycan profiles including modification of all hydroxyl groups in thesample such as permethylation. Such processes require large sampleamounts and produces chemical artefacts such as undermethylatedmolecules lowering the effectivity of the method. These artefact peakscover all minor signals in the spectra, and they can be misinterpretedas glycan structures. It is of importance to note that in glycomeanalyses the important profile to profile differences often reside inthe minor signals. In a specific embodiment the present invention isdirected to site specific modification of the glycans with effectivechemical or enzyme reaction, preferably a quantitative reaction.

Preferred Analytical Technologies for Glycome Analysis MassSpectrometric Analysis of Glycomes

The present invention is specifically directed to quantitative massspectrometric methods for the analysis of glycomes. Most preferred massspectrometric methods are MALDI-TOF mass spectrometry methods.

MALDI-TOF Analysis

The inventors were able to optimise MALDI-TOF mass spectrometry forglycome analysis.

The preferred mass spectrometric analysis process is MALDI-TOF massspectrometry, where the relative signal intensities of the unmodifiedglycan signals represent their relative molar proportions in the sample,allowing relative quantification of both neutral (Naven & Harvey, 19xx)and sialylated (Papac et al., 1996) glycan signals. Preferredexperimental conditions according to the present invention are describedunder Experimental procedures of Examples listed below.

Preferred Mass Ranges for MALDI-TOF Analysis and Released Non-modifiedGlycomes

For MALDI-TOF mass spectrometry of unmodified glycans in positive ionmode, optimal mass spectrometric data recording range according to thepresent invention is over m/z 200, more preferentially between m/z200-10000, or even more preferably between m/z 200-4000 for improveddata quality. In the most preferred form according to the presentinvention, the data is recorded between m/z 700-4000 for accuraterelative quantification of glycan signals.

For MALDI-TOF mass spectrometry of unmodified glycans in negative ionmode, optimal mass spectrometric data recording range according to thepresent invention is over m/z 300, more preferentially between m/z300-10000, or even more preferably between m/z 300-4000 for improveddata quality. In the most preferred forms according to the presentinvention, the data is recorded between m/z 700-4000 or most preferablybetween m/z 800-4000 for accurate relative quantification of glycansignals.

Practical m/z-Ranges

The practical ranges comprising most of the important signals, asobserved by the present invention may be more limited than these.Preferred practical ranges includes lower limit of about m/z 400, morepreferably about m/z 500, and even more preferably about m/z 600, andmost preferably m/z about 700 and upper limits of about m/z 4000, morepreferably m/z about 3500 (especially for negative ion mode), even morepreferably m/z about 3000 (especially for negative ion mode), and inparticular at least about 2500 (negative or positive ion mode) and forpositive ion mode to about m/z 2000 (for positive ion mode analysis).The preferred range depends on the sizes of the sample glycans, sampleswith high branching or polysaccharide content or high sialylation levelsare preferably analysed in ranges containing higher upper limits asdescribed for negative ion mode. The limits are preferably combined toform ranges of maximum and minimum sizes or lowest lower limit withlowest higher limit, and the other limits analogously in order ofincreasing size

Preferred Analysis Modes for MALDI-TOFfor Effective Glycome Analysis

The inventors were able to show effective quantitative analysis in bothnegative and positive mode mass spectrometry.

Sample Handling

The inventors developed optimised sample handling process forpreparation of the samples for MALDI-TOF mass spectrometry.

Glycan Purification

The glycan purification method according to the present inventionconsists of at least one of purification options, preferably in specificcombinations described below, including the following purificationoptions:

1) Precipitation-extraction; 2) Ion-exchange;

3) Hydrophobic interaction;4) Hydrophilic interaction; and5) Affinity to graphitized carbon.1) Precipitation-extraction may include precipitation of glycans orprecipitation of contaminants away from the glycans. Preferredprecipitation methods include:

-   1. Glycan material precipitation, for example acetone precipitation    of glycoproteins, oligosaccharides, glycopeptides, and glycans in    aqueous acetone, preferentially ice-cold over 80% (v/v) aqueous    acetone; optionally combined with extraction of glycans from the    precipitate, and/or extraction of contaminating materials from the    precipitate;-   2. Protein precipitation, for example by organic solvents or    trichloroacetic acid, optionally combined with extraction of glycans    from the precipitate, and/or extraction of contaminating materials    from the precipitate;-   3. Precipitation of contaminating materials, for example    precipitation with trichloroacetic acid or organic solvents such as    aqueous methanol preferentially about 2/3 aqueous methanol for    selective precipitation of proteins and other non-soluble materials    while leaving glycans in solution;    2) Ion-exchange may include ion-exchange purification or enrichment    of glycans or removal of contaminants away from the glycans.    Preferred ion-exchange methods include:-   1. Cation exchange, preferably for removal of contaminants such as    salts, polypeptides, or other cationizable molecules from the    glycans; and-   2. Anion exchange, preferably either for enrichment of acidic    glycans such as sialylated glycans or removal of charged    contaminants from neutral glycans, and also preferably for    separation of acidic and neutral glycans into different fractions.    3) Hydrophilic interaction may include purification or enrichment of    glycans due to their hydrophilicity or specific adsorption to    hydrophilic materials, or removal of contaminants such as salts away    from the glycans. Preferred hydrophilic interaction methods include:-   1. Hydrophilic interaction chromatography, preferably for    purification or enrichment of glycans and/or glycopeptides;-   2. Adsorption of glycans to cellulose in hydrophobic solvents for    their purification or enrichment, preferably to microcrystalline    cellulose, and even more preferably using an    n-butanol:methanol:water or similar solvent system for adsorption    and washing the adsorbed glycans, in most preferred system    n-butanol:methanol:water in relative volumes of 10:1:2, and water or    water:ethanol or similar solvent system for elution of purified    glycans from cellulose.    4) Affinity to graphitized carbon may include purification or    enrichment of glycans due to their affinity or specific adsorption    to graphitized carbon, or removal of contaminants away from the    glycans. Preferred graphitized carbon affinity methods includes    porous graphitized carbon chromatography.

Preferred purification methods according to the invention includecombinations of one or more purification options. Examples of the mostpreferred combinations include the following combinations:

1) For neutral underivatized glycan purification: 1. cation exchange ofcontaminants, 2. hydrophobic adsorption of contaminants, and 3.graphitized carbon affinity purification of glycans.1) For sialylated underivatized glycan purification: 1. cation exchangeof contaminants, 2. hydrophobic adsorption of contaminants, 3.adsorption of glycans to cellulose, and 4. graphitized carbon affinitypurification of glycans.

NMR-analysis of Glycomes

The present invention is directed to analysis of released glycomes byspectrometric method useful for characterization of the glycomes. Theinvention is directed to NMR spectroscopic analysis of the mixtures ofreleased glycans. The inventors showed that it is possible to produce areleased glycome from human stem cells in large scale enough and usefulpurity for NMR-analysis of the glycome.

In a preferred embodiment the NMR-analysis of the stem cell glycome isone dimensional proton NMR-analysis showing structural reporter groupsof the major components in the glycome. The present invention is furtherdirected to combination of the mass spectrometric and NMR analysis ofstem cells.

Preferred Target Cell Populations and Types for Glycome AnalysisAccording to the Invention Early Human Cell Populations Human Stem CellsAnd Multipotent Cells

Under broadest embodiment the present invention is directed to all typesof human stem cells, meaning fresh and cultured human stem cells. Thestem cells according to the invention do not include traditional cancercell lines, which may differentiate to resemble natural cells, butrepresent non-natural development, which is typically due to chromosomalalteration or viral transfection. Stem cells include all types ofnon-malignant multipotent cells capable of differentiating to other celltypes. The stem cells have special capacity stay as stem cells aftercell division, the self-reneval capacity.

Under the broadest embodiment for the human stem cells, the presentinvention describes novel special glycan profiles and novel analytics,reagents and other methods directed to the glycan profiles. Theinvention shows special differences in cell populations with regard tothe novel glycan profiles of human stem cells.

The present invention is further directed to the novel structures andrelated inventions with regard to the preferred cell populationsaccording to the invention. The present invention is further directed tospecific glycan structures, especially terminal epitopes, with regard tospecific preferred cell population for which the structures are new.

Preferred Types of Early Human Cells

The invention is directed to specific types of early human cells basedon the tissue origin of the cells and/or their differentiation status.

The present invention is specifically directed to early human cellpopulations meaning multipotent cells and cell populations derivedthereof based on origins of the cells including the age of donorindividual and tissue type from which the cells are derived, includingpreferred cord blood as well as bone marrow from older individuals oradults. Preferred differentiation status based classification includespreferably “solid tissue progenitor” cells, more preferably“mesenchymal-stem cells”, or cells differentiating to solid tissues orcapable of differentiating to cells of either ectodermal, mesodermal, orendodermal, more preferentially to mesenchymal stem cells.

The invention is further directed to classification of the early humancells based on the status with regard to cell culture and to two majortypes of cell material. The present invention is preferably directed totwo major cell material types of early human cells including fresh,frozen and cultured cells.

Cord Blood Cells, Embryonal-type Cells and Bone Marrow Cells

The present invention is specifically directed to early human cellpopulations meaning multipotent cells and cell populations derivedthereof based on the origin of the cells including the age of donorindividual and tissue type from which the cells are derived.

-   -   a) from early age-cells such 1) as neonatal human, directed        preferably to cord blood and related material, and 2) embryonal        cell-type material    -   b) from stem and progenitor cells from older individuals        (non-neonatal, preferably adult), preferably derived from human        “blood related tissues” comprising, preferably bone marrow        cells.

Cells Differentiating to Solid Tissues, Preferably to Mesenchymal StemCells

The invention is specifically under a preferred embodiment directed tocells, which are capable of differentiating to non-hematopoietictissues, referred as “solid tissue progenitors”, meaning to cellsdifferentiating to cells other than blood cells. More preferably thecell population produced for differentiation to solid tissue are“mesenchymal-type cells”, which are multipotent cells capable ofeffectively differentiating to cells of mesodermal origin, morepreferably mesenchymal stem cells.

Most of the prior art is directed to hematopoietic cells withcharacteristics quite different from the mesenchymal-type cells andmesenchymal stem cells according to the invention.

Preferred solid tissue progenitors according to the invention includesselected multipotent cell populations of cord blood, mesenchymal stemcells cultured from cord blood, mesenchymal stem cells cultured/obtainedfrom bone marrow and embryonal-type cells. In a more specific embodimentthe preferred solid tissue progenitor cells are mesenchymal stem cells,more preferably “blood related mesenchymal cells”, even more preferablymesenchymal stem cells derived from bone marrow or cord blood.

Under a specific embodiment CD34+ cells as a more hematopoietic stemcell type of cord blood or CD34+ cells in general are excluded from thesolid tissue progenitor cells.

Fresh and Cultured Cells Fresh Cells

The invention is especially directed to fresh cells from healthyindividuals, preferably non-modulated cells, and non-manipulated cells.

The invention is in a preferred embodiment directed to “fresh cells”meaning cells isolated from donor and not cultivated in a cell culture.It is realized by the invention that the current cell culture procedureschange the status of the cells. The invention is specifically directedto analysis of fresh cell population because the fresh cellscorresponding closely to the actual status of the individual donor withregard to the cell material and potential fresh cell population areuseful for direct transplantation therapy or are potential raw materialfor production of further cell materials.

The inventors were able to show differences in the preferred fresh cellpopulations derived from early human cells, most preferably from cordblood cells. The inventors were able to produce especially “homogenouscell populations” from human cord blood, which are especially preferredwith various aspects of present invention. The invention is furtherdirected to specific aspects of present invention with regard to cellpurification processes for fresh cells, especially analysis of potentialcontaminations and analysis thereof during the purification of cells.

In a more preferred embodiment the fresh cells are materials relatedto/derived from healthy individuals. The healthy individual means thatthe person is not under treatment of cancer, because such treatmentwould effectively change the status of the cells, in another preferredembodiment the healthy person is receiving treatment of any other majordisease including other conditions which would change the status of thecells.

It is realized that in some cases fresh cells may be needed to beproduced for example for cell transplantation to a cancer patient usingcells previously harvested from such a patient, under a separateembodiment the present invention is further directed to analysis of andother aspects of invention with regard to such cell material.

Non-modulated Cells

Even more preferably the fresh cells are “non-modulated cells” meaningthat the cells have not been modulated in vivo by treatments affectinggrowth factor or cytokine release. For example stem cells may bereleased to peripheral blood by growth factors such as CSF (colonystimulating growth factor). Such treatment is considered to alter thestatus of cells from preferred fresh cells. The modulation may causepermanent changes in all or part of the cells, especially by causingdifferentiation.

Non-manipulated Cells

Even more preferably the fresh cells are “non-manipulated cells” meaningthat the cells have not been manipulated by treatments permanentlyaltering the status of the cells, the permanent manipulation includingalterations of the genetic structure of the cells. The manipulationsinclude gene transfection, viral transduction and induction of mutationsfor example by radiation or by chemicals affecting the geneticstructures of the cells.

Limited Fresh Cells Excluding Certain Specifically SelectedHematopoietic Stem Cell Populations

A more preferred limited group of fresh cells is directed to especiallyto effectively solid tissue forming cells and their precursors. Underspecific embodiment this group does not include specifically selectedmore hematopoietic stem cell like cell populations such as

-   -   a) cell population selected as CD34+ cells from peripheral blood        or bone marrow and    -   b) in another limited embodiment also total bone marrow and        peripheral blood mononuclear cells are excluded.

It is relaized that the fresh cell populations may comprise in part samecells as CD34+ when the cells are not selected with regard to thatmarker. It is realized that exact cell population selected with regardto the marker are not preferred according to the invention as solidtissue forming cells.

Another limited embodiment excludes specifically selected CD34+ cellpopulations from cord blood and/or total mononuclear cells from cordblood. The invention is further directed to limited fresh cellpopulations when all CD34+ cell populations and/or all total cellpopulations of peripheral blood, bone marrow and cord blood areexcluded. The invention is further directed to the limited fresh cellpopulations when CD34+ cell population were excluded, and when bothCD34+ cell populations and all the three total cell populationsmentioned above are excluded.

Cultured Cells

The inventors found specific glycan structures in early human cells, andpreferred subpopulations thereof according to the invention when thecells are cultured. Certain specific structures according to theinvention were revealed especially for cultured cells, and specialalterations of the specific glycans according to the invention wererevealed in cultured cell populations.

The invention revealed special cell culture related reagents, methodsand analytics that can be used when there is risk for by potentiallyharmful carbohydrate contaminations during the cell culture process.

Cultured Modulated Cells

It is further realized that the cultured cells may be modulated in orderto enhance cell proliferation. Under specific embodiment the presentinvention is directed to the analysis and other aspects of the inventionfor cultured “modulated cells”, meaning cells that are modulated by theaction of cytokines and/or growth factors. The inventors note that partof the early changes in cultured cells are related to certain extent tothe modulation.

The present invention is preferably directed to cultured cells, whenthese are non-manipulated. The invention is further directed toobservation of changes induced by manipulation in cell populationsespecially when these are non-intentionally induced by environmentalfactors, such as environmental radiation and potential harmfulmetabolites accumulating to cell preparations.

Preferred Types of Cultured Cells

The present invention is specifically directed to cultured solid tissueprogenitors as preferred cultured cells. More preferably the presentinvention is directed to mesenchymal-type cells and embryonal-type cellsas preferred cell types for cultivation. Even more preferredmesenchymal-type cells are mesenchymal stem cells, more preferablymesenchymal stem cells derived from cord blood or bone marrow.

Under separate embodiment the invention is further directed to culturedhematopoietic stem cells as a preferred group of cultured cells.

Subgroup of Multipotent Cultured Cells

The present invention is especially directed to cultured multipotentcells and cell populations. The preferred multipotent cultured cellmeans various multipotent cell populations enriched in cell cultures.The inventors were able to reveal special characteristics of the stemcell type cell populations grown artificially. The multipotent cellsaccording to the invention are preferably human stem cells.

Cultured Mesenchymal Stem Cells

The present invention is especially directed to mesenchymal stem cells.The most preferred types of mesenchymal stem cells are derived fromblood related tissues, referred as “blood-related mesenchymal cells”,most preferably human blood or blood forming tissue, most preferablyfrom human cord blood or human bone marrow or in a separate embodimentare derived from embryonal type cells. Mesenchymal stem cells derivedfrom cord blood and from bone marrow are preferred separately.

Cultured Embryonal-type Cells and Cell Populations

The inventors were able to reveal specific glycosylation nature ofcultured embryonal-type cells according to the invention. The presentinvention is specifically directed to various embryonal type cells aspreferred cultivated cells with regard to to the present invention.

Early Blood Cell Populations and Corresponding Mesenchymal Stem CellsCord Blood

The early blood cell populations include blood cell materials enrichedwith multipotent cells.

The preferred early blood cell populations include peripheral bloodcells enriched with regard to multipotent cells, bone marrow bloodcells, and cord blood cells. In a preferred embodiment the presentinvention is directed to mesenchymal stem cells derived from early bloodor early blood derived cell populations, preferably to the analysis ofthe cell populations.

Bone Marrow

Another separately preferred group of early blood cells is bone marrowblood cells. These cell do also comprise multipotent cells. In apreferred embodiment the present invention is directed to directed tomesenchymal stem cells derived from bone marrow cell populations,preferably to the analysis of the cell populations.

Preferred Subpopulations of Early Human Blood Cells

The present invention is specifically directed to subpopulations ofearly human cells. In a preferred embodiment the subpopulations areproduced by selection by an antibody and in another embodiment by cellculture favouring a specific cell type. In a preferred embodiment thecells are produced by an antibody selection method preferably from earlyblood cells. Preferably the early human blood cells are cord bloodcells.

The CD34 positive cell population is relatively large and heterogenous.It is not optimal for several applications aiming to produce specificcell products. The present invention is preferably directed tospecifically selected non-CD34 populations meaning cells not selectedfor binding to the CD34-marker, called homogenous cell populations. Thehomogenous cell populations may be of smaller size mononuclear cellpopulations for example with size corresponding to CD133+ cellpopulations and being smaller than specifically selected CD34+ cellpopulations. It is further realized that preferred homogenoussubpopulations of early human cells may be larger than CD34+ cellpopulations.

The homogenous cell population may a subpopulation of CD34+ cellpopulation, in preferred embodiment it is specifically a CD133+ cellpopulation or CD133-type cell population. The “CD133-type cellpopulations” according to the invention are similar to the CD133+ cellpopulations, but preferably selected with regard to another marker thanCD133. The marker is preferably a CD133-coexpressed marker. In apreferred embodiment the invention is directed to CD133+ cell populationor CD133+ subpopulation as CD133-type cell populations. It is realizedthat the preferred homogeneous cell populations further includes othercell populations than which can be defined as special CD133-type cells.

Preferably the homogenous cell populations are selected by binding aspecific binder to a cell surface marker of the cell population. In apreferred embodiment the homogenous cells are selected by a cell surfacemarker having lower correlation with CD34-marker and higher correlationwith CD133 on cell surfaces. Preferred cell surface markers includeα3-sialylated structures according to the present invention enriched inCD133-type cells. Pure, preferably complete, CD133+ cell population arepreferred for the analysis according to the present invention.

The present invention is directed to essential mRNA-expression markers,which would allow analysis or recognition of the cell populations frompure cord blood derived material. The present invention is specificallydirected to markers specifically expressed on early human cord bloodcells.

The present invention is in a preferred embodiment directed to nativecells, meaning non-genetically modified cells. Genetic modifications areknown to alter cells and background from modified cells. The presentinvention further directed in a preferred embodiment to freshnon-cultivated cells.

The invention is directed to use of the markers for analysis of cells ofspecial differentiation capacity, the cells being preferably human bloodcells or more preferably human cord blood cells.

Preferred Purities of the Cell Populations

The preferred purity depends of the affinity of the antibody used. Forpurification using commercial CD34-antibody preferred purity of completecell population is at least 90%, more preferably at least 93%, and mostpreferably at least 95%. In a purification process according toinvention by anti-CD133 antibody preferred purity of complete cellpopulation is at least 90%, more preferably at least 93%, and mostpreferably at least 95%.

The present invention is directed to complete cell populations fromhuman early blood with purity of at least at least 85%, more preferablyat least 90%, even more preferably with increasing preference 91%, 92%,93%, 94%, 95% respectively and most preferably with increasingpreference at least 95%, 96%, 97% or 98%. In a specific embodiment thepresent invention is directed to ultrapure complete cell population inwhich the level of impurities is less than 10%, more preferably lessthan 5% and most preferably less than 3%. The innovation is specificallydirected to complete cell populations purified by anti CD34 andanti-CD133 antibodies.

In a specific embodiment the present invention is directed to highlypurified human complete CD133+ and CD 34+ cell populations derived fromcord blood

Preferred Cord Blood Cell Populations and Characteristics Cord BloodCell Populations

Preferred cord blood cell populations according to the invention includetotal mononuclear cells and subpopulations thereof from cord blood. Thepresent invention is further directed to enriched multipotent cells fromcord blood. In a preferred embodiment the enriched cells are CD133+cells, Lin− (lineage negative) cells, or CD34+ cells from cord blood,even more preferably the enriched cells are CD133+ cells, or Lin−(lineage negative) cells.

In a preferred embodiment the present invention is directed tomesenchymal stem cells derived from cord blood or cord blood derivedcell populations and analysis thereof according to the invention. Apreferred group of mesenchymal stem cells derived from cord blood ismesenchymal stem cells differentiating into cells forming soft tissuessuch as adipose tissue.

Preferred Purity of Reproducibly Highly Purified Mononuclear CompleteCell Populations from Human Cord Blood

The present invention is specifically directed to production of purifiedcell populations from human cord blood. As described above, productionof highly purified complete cell preparations from human cord blood hasbeen a problem in the field. In the broadest embodiment the invention isdirected to biological equivalents of human cord blood according to theinvention, when these would comprise similar markers and which wouldyield similar cell populations when separated similarly as the CD133+cell population and equivalents according to the invention or when cellsequivalent to the cord blood is contained in a sample further comprisingother cell types. It is realized that characteristics similar to thecord blood can be at least partially present before the birth of ahuman. The inventors found out that it is possible to produce highlypurified cell populations from early human cells with purity useful forexact analysis of sialylated glycans and related markers.

Preferred Bone Marrow Cells

The present invention is directed to multipotent cell populations orearly human blood cells from human bone marrow. Most preferred are bonemarrow derived mesenchymal stem cells. In a preferred embodiment theinvention is directed to mesenchymal stem cells differentiating to cellsof structural support function such as bone and/or cartilage.

Embryonal-type Cell Populations

The present invention is specifically directed to methods directed toembryonal-type cell populations, preferably when the use does notinvolve commercial or industrial use of human embryos nor involvedestruction of human embryos. The invention is under a specificembodiment directed to use of embryonal cells and embryo derivedmaterials such as embryonal stem cells, whenever or wherever it islegally acceptable. It is realized that the legislation varies betweencountries and regions.

The present invention is further directed to use of embryonal-related,discarded or spontaneously damaged material, which would not be viableas human embryo and cannot be considered as a human embryo. In yetanother embodiment the present invention is directed to use ofaccidentally damaged embryonal material, which would not be viable ashuman embryo and cannot be considered as human embryo.

It is further realized that early human blood derived from human cord orplacenta after birth and removal of the cord during normal deliveryprocess is ethically uncontroversial discarded material, forming no partof human being.

The invention is further directed to cell materials equivalent to thecell materials according to the invention. It is further realized thatfunctionally and even biologically similar cells may be obtained byartificial methods including cloning technologies.

Mesenchymal Multipotent Cells

The present invention is further directed to mesenchymal stem cells ormultipotent cells as preferred cell population according to theinvention. The preferred mesencymal stem cells include cells derivedfrom early human cells, preferably human cord blood or from human bonemarrow. In a preferred embodiment the invention is directed tomesenchymal stem cells differentiating to cells of structural supportfunction such as bone and/or cartilage, or to cells forming soft tissuessuch as adipose tissue.

Product by Process

The present invention is specifically directed to the glycan fractionproduced according to the present invention from the pico scale stemcell sample according to the present invention. The preferred glycanfraction is essentially devoid of signals of contaminating moleculeswithin the preferred mass range when analysed by MALDI mass spectrometryaccording to the present invention.

The glycome products from stem cells according to present invention areproduced preferably directely from complete human stem cells or membranefractions thereof, more preferably directly from intact cells aseffectively shown in examples. In another preferred embodiment theglycome fractions are cell surface glycomes and produced directly fromsurfaces of complete human stem cells, preferably intact or essentiallyintact human stem cells according to the invention. In anotherembodiment the glycome products according to the invention are produceddirectly from membrane fraction

Preferred Uses of Glycomes and Analysis thereof with Regard to Status ofCells

Search of Novel of Novel Carbohydrate Marker Structures

It is further realized that the analysis of glycome is useful for searchof most effectively altering glycan structures in the early human cellsfor analysis by other methods. The glycome component identified byglycome analysis according to the invention can be furtheranalysed/verified by known methods such as chemical and/or glycosidaseenzymatic degradation(s) and further mass spectrometric analysis and byfragmentation mass apectrometry, the glycan component can be produced inlarger scale by know chromatographic methods and structure can beverified by NMR-spectroscopy.

The other methods would preferably include binding assay using specificlabelled carbohydrate binding agents including especially carbohydratebinding proteins (lectins, antibodies, enzymes and engineered proteinswith carbohydrate binding activity) and other chemicals such as peptidesor aptamers aimed for carbohydrate binding. It is realized that thenovel marker structure can be used for analysis of cells, cell statusand possible effects of contaminats to cell with similar indicativevalue as specific signals of the glycan mass components in glycomeanalysis by mass spectrometry according to the invention.

The invention is especially directed to search of novel carbohydratemarker structures from cell surfaces, preferably by using cell surfaceprofiling methods. The cell surface carbohydrate marker structures wouldbe further preferred for the analysis and/or sorting of cells.

Control of Cell Status and Potential Contaminations by GlycosylationAnalysis Control of Cell Status Contamination/harmful Effect Due toNature of Raw Material for Producing a Cell Population

Species specific, tissue specific, and individual specific differencesin glycan structures are known. The difference between the origin of thecell material and the potential recipient of transplanted material maycause for example immunologic or allergic problems due to glycosylationdifferences. It is further noticed that culture of cells may causechanges in glycosylation. When considering human derived cell materialsaccording to the present invention, individual specific differences inglycosylation are a potent source of harmful effects.

Control of Raw Material Cell Population

The present invention is directed to control of glycosylation of cellpopulations to be used in therapy.

The present invention is specifically directed to control ofglycosylation of cell materials, preferably when

-   -   1) there is difference between the origin of the cell material        and the potential recipient of transplanted material. In a        preferred embodiment there are potential inter-individual        specific differences between the donor of cell material and the        recipient of the cell material. In a preferred embodiment the        invention is directed to animal or human, more preferably human        specific, individual person specific glycosylation differences.        The individual specific differences are preferably present in        mononuclear cell populations of early human cells, early human        blood cells and embryonal type cells. The invention is        preferably not directed to observation of known individual        specific differences such as blood group antigens changes on        erythrocytes.    -   2) There is possibility in variation due to disease specific        variation in the materials. The present invention is        specifically directed to search of glycosylation differences in        the early cell populations according to the present invention        associated with infectious disease, inflammatory disease, or        malignant disease. Part of the inventors have analysed numerous        cancers and tumors and observed similar types glycosylations as        certain glycosylation types in the early cells.    -   3) There is for a possibility of specific inter-individual        biological differences in the animals, preferably humans, from        which the cell are derived for example in relation to species,        strain, population, isolated population, or race specific        differences in the cell materials.    -   4) When it has been established that a certain cell population        can be used for a cell therapy application, glycan analysis can        be used to control that the cell population has the same        characteristics as a cell population known to be useful in a        clinical setting.

Time Dependent Changes During Cultivation of Cells

Furthermore during long term cultivation of cells spontaneous mutationsmay be caused in cultivated cell materials. It is noted that mutationsin cultivated cell lines often cause harmful defects on glycosylationlevel.

It is further noticed that cultivation of cells may cause changes inglycosylation. It is realized that minor changes in any parameter ofcell cultivation including quality and concentrations of variousbiological organic and inorganic molecules, any physical condition suchas temperature, cell density, or level of mixing may cause difference incell materials and glycosylation. The present invention is directed tomonitoring glycosylation changes according to the present invention inorder to observe change of cell status caused by any cell cultureparameter affecting the cells.

The present invention is in a preferred embodiment directed to analysisof glycosylation changes when the density of cells is altered. Theinventors noticed that This has a major impact of the glycosylationduring cell culture.

It is further realized that if there is limitations in genetic ordifferentiation stability of cells, these would increase probability forchanges in glycan structures. Cell populations in early stage ofdifferentiation have potential to produce different cell populations.The present inventors were able to discover glycosylation changes inearly human cell populations.

Differentiation of Cell Lines

The present invention is specifically directed to observe glycosylationchanges according to the present invention when differentiation of acell line is observed. In a preferred embodiment the invention isdirected to methods for observation of differentiation from early humancell or another preferred cell type according to the present inventionto mesodermal types of stem cell

In case there is heterogeneity in cell material this may causeobservable changes or harmful effects in glycosylation.

Furthermore, the changes in carbohydrate structures, even non-harmful orfunctionally unknown, can be used to obtain information about the exactgenetic status of the cells.

The present invention is specifically directed to the analysis ofchanges of glycosylation, preferably changes in glycan profiles,individual glycan signals, and/or relative abundancies of individualglycans or glycan groups according to the present invention in order toobserve changes of cell status during cell cultivation.

Analysis of Supporting/feeder Cell Lines

The present invention is specifically directed to observe glycosylationdifferences according to the present invention, on supporting/feedercells used in cultivation of stem cells and early human cells or otherpreferred cell type. It is known in the art that some cells havesuperior activities to act as a support/feeder cells than other cells.In a preferred embodiment the invention is directed to methods forobservation of differences on glycosylation on these supporting/feedercells. This information can be used in design of novel reagents tosupport the growth of the stem cells and early human cells or otherpreferred cell type.

Contaminations or Alterations in Cells Due to Process ConditionsConditions and Reagents Inducing Harmful Glycosylation or HarmfulGlycosylation Related Effects to Cells During Cell Handling

The inventors further revealed conditions and reagents inducing harmfulglycans to be expressed by cells with same associated problems as thecontaminating glycans. The inventors found out that several reagentsused in a regular cell purification processes caused changes in earlyhuman cell materials.

It is realized, that the materials during cell handling may affect theglycosylation of cell materials. This may be based on the adhesion,adsorption, or metabolic accumulation of the structure in cells underprocessing.

In a preferred embodiment the cell handling reagents are tested withregard to the presence glycan component being antigenic or harmfullstructure such as cell surface NeuGc, Neu-O-Ac or mannose structure. Thetesting is especially preferred for human early cell populations andpreferred subpopulations thereof.

The inventors note effects of various effector molecules in cell cultureon the glycans expressed by the cells if absortion or metabolic transferof the carbohydrate structures have not been performed. The effectorstypically mediate a signal to cell for example through binding a cellsurface receptor.

The effector molecules include various cytokines, growth factors, andtheir signalling molecules and co-receptors. The effector molecules maybe also carbohydrates or carbohydrate binding proteins such as lectins.

Controlled Cell Isolation/purification and Culture Conditions to AvoidContaminations with Harmful Glycans or other Alteration In Glycome Level

Stress Caused by Cell Handling

It is realized that cell handling including isolation/purification, andhandling in context of cell storage and cell culture processes are notnatural conditions for cells and cause physical and chemical stress forcells. The present invention allows control of potential changes causedby the stress. The control may be combined by regular methods may becombined with regular checking of cell viability or the intactness ofcell structures by other means.

Examples of Physical and/or Chemical Stress in Cell Handling Step

Washing and centrifuging cells cause physical stress which may break orharm cell membrane structures. Cell purifications and separations oranalysis under non-physiological flow conditions also expose cells tocertain non-pnysiological stress. Cell storage processes and cellpreservation and handling at lower temperatures affects the membranestructure. All handling steps involving change of composition of mediaor other solution, especially washing solutions around the cells affectthe cells for example by altered water and salt balance or by alteringconcentrations of other molecules effecting biochemical andphysiological control of cells.

Observation and Control of Glycome Changes by Stress in Cell HandlingProcesses

The inventors revealed that the method according to the invention isuseful for observing changes in cell membranes which usually effectivelyalter at least part of the glycome observed according to the invention.It is realized that this related to exact organization and intactstructures cell membranes and specific glycan structures being part ofthe organization.

The present invention is specifically directed to observation of totalglycome and/or cell surface glycomes, these methods are further aimedfor the use in the analysis of intactness of cells especially in contextof stressfull condition for the cells, especially when the cells areexposed to physical and/or chemical stress. It is realized that each newcell handling step and/or new condition for a cell handling step isuseful to be controlled by the methods according to the invention. It isfurther realized that the analysis of glycome is useful for search ofmost effectively altering glycan structures for analysis by othermethods such as binding by specific carbohydrate binding agentsincluding especially carbohydrate binding proteins (lectins, antibodies,enzymes and engineered proteins with carbohydrate binding activity).

Controlled Cell Preparation (Isolation or Purification) with Retard toReagents

The inventors analysed process steps of common cell preparation methods.Multiple sources of potential contamination by animal materials werediscovered.

The present invention is specifically directed to carbohydrate analysismethods to control of cell preparation processes. The present inventionis specifically directed to the process of controlling the potentialcontaminations with animal type glycans, preferably N-glycolylneuraminicacid at various steps of the process.

The invention is further directed to specific glycan controlled reagentsto be used in cell isolation

The glycan-controlled reagents may be controlled on three levels:

-   -   1. Reagents controlled not to contain observable levels of        harmful glycan structure, preferably N-glycolylneuraminic acid        or structures related to it    -   2. Reagents controlled not to contain observable levels of        glycan structures similar to the ones in the cell preparation    -   3. Reagent controlled not to contain observable levels of any        glycan structures.

The control levels 2 and 3 are useful especially when cell status iscontrolled by glycan analysis and/or profiling methods. In case reagentsin cell preparation would contain the indicated glycan structures thiswould make the control more difficult or prevent it. It is furthernoticed that glycan structures may represent biological activitymodifying the cell status.

Cell Preparation Methods Including Glycan-controlled Reagents

The present invention is further directed to specific cell purificationmethods including glycan-controlled reagents.

Preferred Controlled Cell Purification Process

The present invention is especially directed to controlled production ofhuman early cells containing one or several following steps. It wasrealized that on each step using regular reagents in following processthere is risk of contamination by extragenous glycan material. Theprocess is directed to the use of controlled reagents and materialsaccording to the invention in the steps of the process.

Preferred purification of cells includes at least one of the stepsincluding the use of controlled reagent, more preferably at least twosteps are included, more preferably at least 3 steps and most preferablyat least steps 1, 2, 3, 4, and 6.

-   -   1. Washing cell material with controlled reagent.    -   2. When antibody based process is used cell material is in a        preferred embodiment blocked with controlled Fc-receptor        blocking reagent. It is further realized that part of        glycosylation may be needed in a antibody preparation, in a        preferred embodiment a terminally depleted glycan is used.    -   3. Contacting cells with immobilized cell binder material        including controlled blocking material and controlled cell        binder material. In a more preferred the cell binder material        comprises magnetic beads and controlled gelatin material        according the invention. In a preferred embodiment the cell        binder material is controlled, preferably a cell binder antibody        material is controlled. Otherwise the cell binder antibodies may        contain even N-glycolylneuraminic acid, especially when the        antibody is produced by a cell line producing        N-glycolylneuraminic acid and contaminate the product.    -   4. Washing immobilized cells with controlled protein preparation        or non-protein preparation.    -   In a preferred process magnetic beads are washed with controlled        protein preparation, more preferably with controlled albumin        preparation.    -   5. Optional release of cells from immobilization.    -   6. Washing purified cells with controlled protein preparation or        non-protein preparation.

In a preferred embodiment the preferred process is a method usingimmunomagnetic beads for purification of early human cells, preferablypurification of cord blood cells.

The present invention is further directed to cell purification kit,preferably an immunomagnetic cell purification kit comprising at leastone controlled reagent, more preferably at least two controlledreagents, even more preferably three controlled reagents, evenpreferably four reagents and most preferably the preferred controlledreagents are selected from the group: albumin, gelatin, antibody forcell purification and Fc-receptor blocking reagent, which may be anantibody.

Storage Induced Changes Causing Harmful Glycosylations or Change in theStatus of Cells

It was realized that storage of the cell materials may cause harmfulchanges in glycosylation or changes in cell status observable byglycosylation analysis according to the present invention.

Changes Observable in Context of Low Temperature Storage or Handling ofCells

The inventors discovered that keeping the cells in lower temperaturesalters the status of cells and this observable analysing the chemicalstructures of cells, preferably the glycosylation of the cells. Thelower temperatures usually vary between 0-36 degrees of Celsiusincluding for example incubator temperature below about 36 degrees ofCelsius more preferably below 35 degrees of Celsius, various roomtemperatures, cold room and fridge temperatures typically between 2-10degrees of Celsius, and temperatures from incubation on ice close to 0degrees of Celsius typically between 0-4 degrees of Celsius. The loweredtemperatures are typically needed for processing of cells or temporarystorage of the preferred cells.

The present invention is specifically directed to analysis of the statusof cells kept in low temperatures in comparison to natural bodytemperatures. In a preferred embodiment the control is performed aftercertain time has passed from process in lower temperature in order toconfirm the recovery of the cells from the lower temperature. In anotherpreferred embodiment the present invention is directed to development oflower temperature methods by controlling the chemical structures ofcells, preferably by controlling glycosylation according to the presentinvention.

Changes Observable in Context of Cryopreservation

The inventors discovered that cryopreservation alters the status ofcells and this observable analysing the chemical structures of cells,preferably the glycosylation of the cells. The present invention isspecifically directed to analysis of the status of cryopreserved cells.In a preferred embodiment the control is performed after certain timehas passed from preservation in order to confirm the recovery of thecells from the cryopreservation. In another preferred embodiment thepresent invention is directed to development of cryopreservetion methodsby controlling the chemical structures of cells, preferably bycontrolling glycosylation according to the present invention.

Contaminations with Harmful Glycans Such as Antigenic Animal TypeGlycans

Several glycans structures contaminating cell products may weaken thebiological activity of the product.

The harmful glycans can affect the viability during handling of cells,or viability and/or desired bioactivity and/or safety in therapeutic useof cells.

The harmful glycan structures may reduce the in vitro or in vivoviability of the cells by causing or increasing binding of destructivelectins or antibodies to the cells. Such protein material may beincluded e.g. in protein preparations used in cell handling materials.Carbohydrate targeting lectins are also present on human tissues andcells, especially in blood and endothelial surfaces. Carbohydratebinding antibodies in human blood can activate complement and causeother immune responses in vivo. Furthermore immune defence lectins inblood or leukocytes may direct immune defence against unusual glycanstructures.

Additionally harmful glycans may cause harmful aggregation of cells invivo or in vitro. The glycans may cause unwanted changes indevelopmental status of cells by aggregation and/or changes in cellsurface lectin mediated biological regulation.

Additional problems include allergenic nature of harmful glycans andmisdirected targeting of cells by endothelial/cellular carbohydratereceptors in vivo.

Contaminations from Reagents

The present invention is specifically directed to control of thereagents used to prevent contamination by harmful glycan structures. Theharmful glycan structures may originate from reagents used during cellhandling processes such as cell preservation, cell preparation, and cellculture.

Preferred reagents to be controlled according to the present inventioninclude cell culture reagents, cell blocking reagents, such as antibodyreceptor blocking reagents, washing solutions during cell processing,material blocking reagents, such as blocking reagents for materials likefor example magnetic beads. Preferably the materials are controlled:

-   -   1. so that these would not contain a contaminating structure,        preferably a NeuGc-structure according to the invention, or more        specifically preferred glycan structure according to the        invention    -   2. so that the materials contain very low amounts or do not        contain any potentially harmful structures according to the        invention.

Abbreviations and Definitions Modification Definitions:

Ac=acetyl ester or acetyl amide modification (C₂H₂O).

S/P or SP=sulphate (SO₃) or phosphate (PO₃H) ester modification, oranother modification of corresponding mass.

Other modifications (Mod)=any modification to the monosaccharide andmodification compositions, either affecting the proposed structure andits molecular mass positively, such as H, H₂, or Pr (propyl, C₃H₇), oraffecting the proposed structure and its molecular mass negatively, suchas —H₂O or —Ac (without acetyl, —C₂H₂O); the latter option correspondingto e.g. proposed elimination products.

Ionized Forms:

In mass spectrometry, glycans occur in ionized forms such as [M+Na]⁺,[M+K]⁺, [M-H]⁻, or [M-2H+Na]⁻. The present invention is directed tofinding out proposed monosaccharide and modification compositions formass spectrometric signals, based on most probable combinations ofmonosaccharides and modifications, typically according to definitionslisted above and preferably based on sample type-specific monosaccharideand modification selections such as those listed in the Examples andTables below. Single monosaccharide and modification compositionspotentially give rise to multiple mass spectrometric signals, forexample [M+Na]⁺ and [M+K]⁺ adduct ions, and the present invention isespecially directed to taking this phenomenon into account in theanalysis results.

Molecular Mass and m/z Calculations, and Abbreviations Used in the Text:

Molecular masses and m/z values for proposed monosaccharide compositionsand ionized forms therefrom can be calculated from the correspondingatom compositons according to common knowledge of the art.

In the following text, figures, and tables the m/z values of proposedmonosaccharide compositions may be expressed as the m/z value of thefirst isotope and rounded down for clarity. The corresponding moreprecise expressions can be derived from the proposed compositions and/orexperimental data, and they are optionally, especially when needed forinterpretation of the analysis results, expressed with more precision inthe text, tables, and/or figures.

Preferred Forms of Monosaccharide and Modification Compositions:

In analyses of human early cells or biological reagents or biologicalsamples occurring in context of human early cell analysis, preferredmonosaccharide and modification combinations according to the presentinventions include those listed in the Examples and Tables below.

Structural Features Derived from the Glycome Compositions

Marker Structures and Glycomes

The invention revealed individual glycan structures and structuregroups, which are novel markers for the cell materials according to theinvention. The present invention is directed to the use of the markerstructures and their combinations for analysis, for labelling and forcell separation, as modification targets and for other methods accordingto invention.

The present invention revealed large groups of glycans, which can bederived from cells according to the invention The present invention isespecially directed to release of various protein or lipid linkedoligosaccharide and/or polysaccharide chains as free glycan, glycanreducing end derivative or glycopeptide freactions referred as glycomesfrom the cell material according to the invention The glycans can bereleased separately from differently linked glycan groups on proteinsand or glycolipids or in combined process producing several isolatedglycome fractions and/or combined glycome fractions, which compriseglycans released at least from two different glycomes. The relativeamounts of various components/component groups observable in glycanprofiling as peaks in mass spectra and in quantitative presentations ofglycan based profiling information, especially in analysis of massspectrometric and/or NMR-data were revealed to be characteristic forindividual cell types. The glycomes was further revealed to containglycan subgroups or subglycomes which are very useful forcharacterization of the cell materials according to the invention.

Glycome Types Based on Linkage Structures

The invention revealed four major glycome types based on the linkagestructures. Two protein linked glycomes are N-linked glycomes andO-linked glycomes. The majority of the glycosaminoglycan (gag) glycomes(gagomes) are also linked to certain proteins by specific core andlinkage structures. The glycolipid glycome is linked to lipids, usuallysphingolipids.

Core Structures of Glycomes and Terminal Glycome Specific and CommonStructures

The invention has revealed specific glycan core structures for thespecific subglycomes studied. The various structures in specificglycomes were observed to contain common reducing end core structuressuch as N-glycan and O-glycan, Glycosaminoglycan and glycolipid cores.The cores are elongated with varying glycan chains usually comprisinggroups of glycans with different chain length. The presence of a corestructures is often observably as a characteristic monosaccharidecomposition as monosaccharide composition of the core structure causingdifferent relation of monosaccharide residues in speficic glycan signalsof glycomes when profiled by mass spectrometry according to theinvention. The present invention further revealed specific non-reducingend terminal structures of specific marker glycans. Part of thenon-reducing end terminal structures are characteristic for severalglycomes, for example N-acetylactosamine type terminal structures,including fucosylated and sialylated variants were revealed from complexN-glycans, O-glycan and Glycolipid glycomes. Part of the structures arespecific for glycomes such terminal Man-structures in Low-mannose andHigh-mannose N-glycans.

Combined Analysis of Different Glycomes

The invention revealed similar structures on protein and lipid linkedglycomes in the cell materials according to the invention. It wasrevealed that combined analysis of the different glycomes is usefulcharacterization of specific cell materials according to the invention.The invention specifically revealed similar lactosamine type structuresin glycolipid and glycoprotein linked glycomes.

The invention further revealed glycosaminoglycan glycome and glycomeprofile useful for the analysis of the cell status and certainsynergistic characteristics glycosaminoglycan glycomes and other proteinlinked glycomes such as non-sialic acid containing acidic structures inN-liked glycomes. The biological roles of glycosaminoglycans andglycolipids in regulation of cell biology and their biosyntheticdifference and distance revealed by glycome analysis make these a usefulcombination for analysis of cell status. It is further realized thatcombination of all all glycomes including O-glycan and N-glycanglycomes, glycolipid glycome and glycosaminoglycan glycome are usefulfor analysis of cells according to the invention. The invention furtherrevealed common chemical structural features in the all glycomesaccording invention supporting the effective combined production,purification and analysis of glycomes according to the invention.

In a preferred embodiment the invention is directed to combined analysisof following glycome combinations, more preferably the glycomes areanalysed from same sample to obtain exact information about the statusof the cell material:

-   -   1. Two protein linked glycomes: N-glycan and O-glycan glycomes    -   2. Glycolipid glycomes with protein linked glycomes, especially        preferred glycolipid glycomes and N-glycan glycomes    -   3. Protein linked glycome or glycomes with glycosaminoglycan        glycome, in preferred embodiment a glycosaminoglycan glycome and        N-glycan glycome.    -   4. Lipid linked glycome or glycomes with glycosaminoglycan        glycome    -   5. Protein linked O-glycan and N-glycan glycomes, glycolipid        glycome and glycosaminoglycan glycome.

The invention further revealed effective methods for the analysis ofdifferent glycomes. It was revealed that several methods developed forsample preparation are useful for both lipid and protein linkedglycomes, in a preferred embodiment proteolytic treatment is used forboth production of protein linked glycome and a lipid linked glycome,especially for production of cell surface glycomes. For production ofTotal cell glycomes according to the invention the extraction ofglycolipids is preferably used for degradation of cells and proteinfraction obtained from the lipid extraction is used for protein linkedglycome analysis. The invention is further directed to the chemicalrelease of glycans, preferably for simultaneous release of both O-linkedand N-linked glycans. Glycolipid and other glycomes, especially N-linkedglycome, can be effectively released enzymatically, the invention isdirected to sequential release of glycans by enzymes, preferablyincluding step of inactivating enzymes between the treatments and usingglycan controlled enzymes to avoid contamination or controllingcontamination of glycans originationg from enzymes.

Common Structural Features of All Glycomes and Preferred CommonSubfeatures

The present invention reveals useful glycan markers for stem cells andcombinations thereof and glycome compositions comprising specificamounts of key glycan structures. The invention is furthermore directedto specific terminal and core structures and to the combinationsthereof.

The preferred glycome glycan structure(s) and/or glycomes from cellsaccording to the invention comprise structure(s) according to theformula C0:

R₁Hexβz{R₃}_(n1)Hex(NAc)_(n2)XyR2,

Wherein X is glycosidically linked disaccharide epitope β4(Fucα6)_(n)GN,wherein n is 0 or 1, or X is nothing and

Hex is Gal or Man or GlcA, HexNAc is GlcNAc or GalNAc,

y is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon,z is linkage position 3 or 4, with the provision that when z is 4 thenHexNAc is GlcNAc and then Hex is Man or Hex is Gal or Hex is GlcA, andwhen z is 3 then Hex is GlcA or Gal and HexNAc is GlcNAc or GalNAc;n1 is 0 or 1 indicating presence or absence of R3;

n2 is 0 or 1, indicating the presence or absence of NAc, with theproviso that n2 can be 0 only when Hexβz is Galβ4, and n2 is preferably0, n2 structures are preferably derived from glycolipids;

R₁ indicates 1-4, preferably 1-3, natural type carbohydrate substituentslinked to the core structures or nothing;R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagine N-glycoside aminoacids and/or peptides derived fromprotein, or natural serine or threonine linked O-glycoside derivativesuch as serine or threonine linked O-glycosides including asparagineN-glycoside aminoacids and/or peptides derived from protein, or when n2is 1 R2 is nothing or a ceramide structure or a derivetive of a ceramidestructure, such as lysolipid and amide derivatives thereof;R3 is nothing or a branching structure respesenting a GlcNAcβ6 or anoligosaccharide with GlcNAcβ6 at its reducing end linked to GalNAc (whenHexNAc is GalNAc); or when Hex is Gal and HexNAc is GlcNAc, and when zis 3 then R3 is Fucα4 or nothing, and when z is 4 R3 is Fucα3 ornothing.

The preferred disaccharide epitopes in the glycan structures andglycomes according to the invention include structures Galβ4GlcNAc,Manβ4GlcNAc, GlcAβ4GlcNAc, Galβ3GlcNAc, Galβ3GalNAc, GlcAβ3GlcNAc,GlcAβ3GalNAc, and Galβ4Glc, which may be further derivatized fromreducing end carbon atom and non-reducing monosaccharide residues and isin a separate embodiment branched from the reducing end residue.Preferred branched epitopes include Galβ4(Fucα3)GlcNAc,Galβ3(Fucα4)GlcNAc, and Galβ3(GlcNAcβ6)GalNAc, which may be furtherderivatized from reducing end carbon atom and non-reducingmonosaccharide residues.

Preferred Epitopes for Methods According to the InventionN-acetyllactosamine Galβ3/4GlcNAc Terminal Epitopes

The two N-acetyllactosamine epitopes Galβ4GlcNAc and/or Galβ3GlcNAcrepresent preferred terminal epitopes present on stem cells or backbonestructures of the preferred terminal epitopes for example furthercomprising sialic acid or fucose derivatisations according to theinvention. In a preferred embodiment the invention is direted tofucosylated and/or non-substituted glycan non-reducing end forms of theterminal epitopes, more preferably to fucosylated and non-substutitutedforms. The invention is especially directed to non-reducing end terminal(non-substituted) natural Galβ4GlcNAc and/or Galβ3GlcNAc-structures fromhuman stem cell glycomes. The invention is in a specific embodimentdirected to non-reducing end terminal fucosylated natural Galβ4GlcNAcand/or Galβ3GlcNAc-structures from human stem cell glycomes.

Preferred Fucosylated N-acetyllactosamines

The preferred fucosylated epitopes are according to the Formula TF:

(Fucα2)_(n1)Galβ3/4(Fucα4/3)_(n2)GlcNAcβ-R

Wherein

n1 is 0 or 1 indicating presence or absence of Fucα2;n2 is 0 or 1, indicating the presence or absence of Fucα4/3 (branch),andR is the reducing end core structure of N-glycan, O-glycan and/orglycolipid.

The preferred structures thus include type 1 lactosaamines (Galβ3GlcNAcbased):

Galβ3(Fucα4)GlcNAc (Lewis a), Fucα2Galβ3GlcNAc H-type 1, structure and,Fucα2Galβ3(Fucα4)GlcNAc (Lewis b) andtype 2 lactosamines (Galβ4GlcNAc based):Galβ4(Fucα3)GlcNAc (Lewis x), Fucα2Galβ4GlcNAc H-type 2, structure and,Fucα2Galβ4(Fucα3)GkcNAc (Lewis y).

The type 2 lactosamines (fucosylated and/or terminal non-substituted)form an especially preferred group in context of embryonal-type stemcells and differentiated cells derived directly from these. Type 1lactosamines (Galβ3GlcNAc—structures) are especially preferred incontext of adult stem cells.

Lactosamines Galβ3/4GlcNAc and Glycolipid Structures Comprising LactoseStructures (Galβ4Glc)

The lactosamines form a preferred structure group with lactose-basedglycolipids. The structures share similar features as products ofβ3/4Gal-transferases. The β3/4 galactose based structures were observedto produce characteristic features of protein linked and glycolipidglycomes.

The invention revealed that furthermore Galβ3/4GlcNAc-structures are akey feature of differentiation releated structures on glycolipids ofvarious stem cell types. Such glycolipids comprise two preferredstructural epitopes according to the invention. The most preferredglycolipid types include thus lactosylceranide based glycosphingolipidsand especially lacto-(Galβ3GlcNAc), such as

lactotetraosylceramide Galβ3GlcNAcβ3Galβ4GlcβCer, preferred structuresfurther including its non-reducing terminal structures selected from thegroup: Galβ3(Fucα4)GlcNAc (Lewis a), Fucα2Galβ3GlcNAc (H-type 1),structure and, Fucα2Galβ3(Fucα4)GlcNAc (Lewis b) or sialylated structureSAα3Galβ3GlcNAc or SAα3Galβ3(Fucα4)GlcNAc, wherein SA is a sialic acid,preferably Neu5Ac preferably replacing Galβ3GlcNAc oflactotetraosylceramide and its fucosylated and/or elogated variants suchas preferably according to the Formula:

(Sacα3)_(n5)(Fucα2)_(n1)Galβ3(Fucα4)_(n3)GlcNAcβ3[Galβ3/4(Fucα4/3)_(n2)GlcNAcβ3]_(n4)Galβ4GlcβCer

whereinn1 is 0 or 1, indicating presence or absence of Fucα2;n2 is 0 or 1, indicating the presence or absence of Fucα4/3 (branch),n3 is 0 or 1, indicating the presence or absence of Fucα4 (branch)n4 is 0 or 1, indicating the presence or absence of (fiicosylated)N-acetyllactosamine elongation;n5 is 0 or 1, indicating the presence or absence of Sacα3 elongation;Sac is terminal structure, preferably sialic acid, with α3-linkage, withthe proviso that when Sac is present, n5 is 1, then n1 is 0 andneolacto (Galβ4GlcNAc)-comprising glycolipids such asneolactoteraosylceramide Galβ4GlcNAcβ3Galβ4GlcβCer, preferred structuresfurther including its non-reducing terminal Galβ4(Fucα3)GlcNAc (Lewisx), Fucα2Galβ4GlcNAc H-type 2, structure and, Fucα2Galβ4(Fucα3)GlcNAc(Lewis y) andits fucosylated and/or elogated variants such as preferably

(Sacα3/6)_(n5)(Fucα2)_(n1)Galβ4(Fucα3)_(n3)GlcNAcβ3[Galβ4(Fucα3)_(n2)GlcNAcβ3]_(n4)Galβ4GlcβCer

n1 is 0 or 1 indicating presence or absence of Fucα2;n2 is 0 or 1, indicating the presence or absence of Fucα3 (branch),n3 is 0 or 1, indicating the presence or absence of Fucα3 (branch)n4 is 0 or 1, indicating the presence or absence of (fucosylated)N-acetyllactosamine elongation,n5 is 0 or 1, indicating the presence or absence of Sacα3/6 elongation;Sac is terminal structure, preferably sialic acid (SA) with α3-linkage,or sialic acid with α6-linkage, with the proviso that when Sac ispresent, n5 is 1, then n1 is 0, and when sialic acid is bound byα6-linkage preferably also n3 is 0.

Preferred Stem Cell Glycosphingolipid Glycan Profiles, Compositions, andMarker Structures

The inventors were able to describe stem cell glycolipid glycomes bymass spectrometric profiling of liberated free glycans, revealing about80 glycan signals from different stern cell types. The proposedmonosaccharide compositions of the neutral glyeans were composed of 2-7Hex, 0-5 HexNAc, and 0-4 dhex. The proposed monosaccharide compositionsof the acidic glycan signals were composed of 0-2 NeuAc, 2-9 Hex, 0-6HexNAc, 0-3 dhex, and/or 0-1 sulphate or phosphate esters. The presentinvention is especially directed to analysis and targeting of such stemcell glycan profiles and/or structures for the uses described in thepresent invention with respect to stem cells.

The present invention is further specifically directed toglycosphingolipid glycan signals specific tostem cell types as describedin the Examples. In a preferred embodiment, glycan signals typical tohESC, preferentially including 876 and 892 are used in their analysis,more preferentially FucHexHexNAcLac, wherein α1,2-Fuc is preferential toα1,3/4-Fuc, and Hex₂HexNAc₁Lac, and more preferentially toGalβ3-[Hex₁HexNAc₁]Lac. In another preferred embodiment, glycan signalstypical to MSC, especially CB MSC, preferentially including 1460 and1298, as well as large neutral glycolipids, especially Hex₂₋₃HexNAc₃Lac,more preferentially poly-N-acetyllactosamine chains, even morepreferentially β1,6-branched, and preferentially terminated with type IILacNAc epitopes as descrbed above, are used in context of MSC accordingto the uses described in the present invention.

Terminal glycan epitopes that were demonstrated in the presentexperiments in stem cell glycosphingolipid glycans are useful inrecognizing stem cells or specifically binding to the stem cells viaglycans, and other uses according to the present invention, includingterminal epitopes: Gal, Galβ4Glc (Lac), Galβ4GlcNAc (LacNAc type 2),Galβ3, Non-reducing terminal HexNAc, Fuc, α1,2-Fuc, α1,3-Fuc, Fucα2Gal,Fucα2Galβ4GlcNAc (H type 2), Fucα2Galβ4Glc (2′-fucosyllactose),Fucα3GlcNAc, Galβ4(Fucα3)GlcNAc (Lex), Fucα3Glc, Galβ4(Fucα3)Glc(3-fucosyllactose), Neu5Ac, Neu5Acα2,3, and Neu5Acα2,6. The presentinvention is further directed to the total terminal epitope profileswithin the total stem cell glycosphingolipid glycomes and/or glycomes.

The inventors were further able to characterize in hESC thecorresponding glycan signals to SSEA-3 and SSEA-4 developmental relatedantigens, as well as their molar proportions within the stem cellglycome. The invention is further directed to quantitative analysis ofsuch stem cell epitopes within the total glycomes or subglycomes, whichis useful as a more efficient alternative with respect to antibodiesthat recognize only surface antigens. In a further embodiment thepresent invention is directed to finding and characterizing theexpression of cryptic developmental and/or stem cell antigens within thetotal glycome profiles by studying total glycan profiles, asdemonstrated in the Examples for α1,2-fucosylated antigen expression inhESC in contrast to SSEA-1 expression in mouse ES cells.

The present invention revealed characteristic variations (increased ordecreased expression in comparision to similar control cell or acontaminatiog cell or like) of both structure types in various cellmaterials according to the invention. The structures were revealed withcharacteristic and varying expression in three different glycome types:N-glycans, O-glycans, and glycolipids. The invention revealed that theglycan structures are a charateristic feature of stem cells and areuseful for various analysis methods according to the invention. Amountsof these and relative amounts of the epitopes and/or derivatives variesbetween cell lines or between cells exposed to different conditionsduring growing, storage, or induction with effector molecules such ascytokines and/or hormones.

The preferred glycome glycan structure(s) and/or glycomes from cellsaccording to the invention comprise structure(s) according to theformula C1:

R₁Hexβz{R₃}_(n1)HexNAcXyR₂,

Wherein X is glycosidically linked disaccharide epitope β4(Fucα6)_(n)GN,wherein n is 0 or 1, or X is nothing and

Hex is Gal or Man or GlcA, HexNAc is GlcNAc or GalNAc,

y is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon,z is linkage position 3 or 4, with the provision that when z is 4 thenHexNAc is GlcNAc and then Hex is Man or Hex is Gal or Hex is GlcA, andwhen z is 3 then Hex is GlcA or Gal and HexNAc is GlcNAc or GalNAc,R₁ indicates 1-4, preferably 1-3, natural type carbohydrate substituentslinked to the core structures,R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacids and/or peptides derivedfrom protein, or natural serine or threonine linked O-glycosidederivative such as serine or threonine linked O-glycosides includingasparagines N-glycoside aminoacids and/or peptides derived from protein.R3 is nothing or a branching structure respesenting a GlcNAcβ6 or anoligosaccharide with GlcNAcβ6 at its reducing end linked to GalNAc (whenHexNAc is GalNAc) or when Hex is Gal and HexNAc is GlcNAc the then whenz is 3 R3 is Fucα4 or nothing and when z is 4 R3 is Fucα3 or nothing.

The preferred disaccharide epitopes in the glycan structures andglycomes according to the invention include structures Galβ4GlcNAc,Manβ4GlcNAc, GlcAβ4GlcNAc, Galβ3GlcNAc, Galβ3GalNAc, GlcAβ3GlcNAc andGlcAβ3GalNAc, which may be further derivatized from reducing end carbonatom and non-reducing monosaccharide residues and is separate embodimentbranched from the reducing end residue. Preferred branched epitopesinclude Galβ4(Fucα3)GlcNAc, Galβ3(Fucα4)GlcNAc, Galβ3(GlcNAcβ6)GalNAc,which may be further derivatized from reducing end carbon atom andnon-reducing monosaccharide residues.

The preferred disaccharide epitopes of glycoprotein or glycolipidstructures present on glycans of human cells according to the inventioncomprise structures based on the formula C2:

R₁Hexβ4GlcNAcXyR₂,

Wherein Hex is Gal OR Man and when Hex is Man then X is glycosidicallylinked disaccharide epitope β4(Fucα6)_(n)GN, wherein n is 0 or 1, or Xis nothing and when Hex is Gal then X is β3GalNAc of O-glycan core orβ2/4/6Manα3/6 terminal of N-glycan core (as in formula NC3)

y is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon,R₁, indicates 1-4, preferably 1-3, natural type carbohydratesubstituents linked to the core structures,when Hex is Gal preferred R1 groups include structures SAα3/6,SAα3/6Galβ4GlcNAcβ3/6,when Hex is Man preferred R1 groups include Manα3, Manα6, branchedstructure Manα3 {Manα6} and elongated variants thereof as described forlow mannose, high-mannose and complex type N-glycans below,R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacids and/or peptides derivedfrom protein, or natural serine or threonine linked O-glycosidederivative such as serine or threonine linked O-glycosides includingasparagines N-glycoside aminoacids and/or peptides derived from protein.

Structures of N-linked Glycomes Common Core Structure of N-linkedGlycomes

The inventors revealed that the N-glycans released by specific N-glycanrelease methods from the cells according to the invention, and preferredcells according to the invention, comprise mostly a specific type ofN-glycan core structure.

The preferred N-glycan structure of each cell type is characterised andrecognized by treating cells with a N-glycan releasing enzyme releasingpractically all N-glycans with core type according to the invention. TheN-glycan relasing enzyme is preferably protein N-glycosidase enzyme,preferably by protein N-glycosidase releasing effectively the N-glycomesaccording to the invention, more preferably protein N-glycosidase withsimilar specificity as protein N-glycosidase F, and in a specificallypreferred embodiment the enzyme is protein N-glycosidase F from F.meningosepticum. Alternative chemical N-glycan release method was usedfor controlling the effective release of the N-glycomes by the N-glycanrelasing enzyme.

The inventors used the NMR glycome analysis according to the inventionfor further characterization of released N-glycomes from small cellsamples available. NMR spectroscopy revealed the N-glycan core signalsof the preferred N-glycan core type of the cells according to theinvention.

The Minimum Formula

The present invention is directed to glycomes derived from stem cellsand comprising a common N-glycosidic core structures. The invention isspecifically directed to minimum formulas covering both GN₁-glycomes andGN₂-glycomes with difference in reducing end structures.

The minimum core structure includes glycans from which reducing endGlcNAc or Fucα6GlcNAc has been released. These are referred asGN₁-glycomes and the components thereof as GN₁-glycans. The presentinvention is specifically directed to natural N-glycomes from human stemcells comprising GN₁-glycans. In a preferred embodiment the invention isdirected to purified or isolated practically pure natural GN₁-glycomefrom human stem cells. The release of the reducing end GlcNAc-unitcompletely or partially may be included in the production of theN-glycome or N-glycans from stem cells for analysis.

The glycomes including the reducing end GlcNAc or Fucα6GlcNAc arereferred as GN₂-glycomes and the components thereof as GN₂-glycans. Thepresent invention is also specifically directed to natural N-glycomesfrom human stem cells comprising GN₂-glycans. In a preferred embodimentthe invention is directed to purified or isolated practically purenatural GN₂-glycome from human stem cells.

The preferred N-glycan core structure(s) and/or N-glycomes from stemcells according to the invention comprise structure(s) according to theformula NC1:

R₁Mβ4GNXyR₂,

Wherein X is glycosidically linked disaccharide epitope β4(Fucα6)_(n)GN,wherein n is 0 or 1, or X is nothing and

y is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon, andR1 indicates 1-4, preferably 1-3, natural type carbohydrate substituentslinked to the core structures,R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacids and/or peptides derivedfrom protein.

It is realized that when the invention is directed to a glycome, theformula indicates mixture of several or typically more than ten or evenhigher number of different structures according to the Formulasdescribing the glycomes according to the invention.

The possible carbohydrate substituents R₁ comprise at least one mannose(Man) residue, and optionally one or several GlcNAc, Gal, Fuc, SAand/GalNAc residues, with possible sulphate and or phosphatemodifications.

When the glycome is released by N-glycosidase the free N-glycomesaccharides comprise in a preferred embodiment reducing end hydroxylwith anomeric linkage A having structure α and/or β, preferably both αand β. In another embodiment the glycome is derivatized by a molecularstructure which can be reacted with the free reducing end of a releasedglycome, such as amine, aminooxy or hydrazine or thiol structures. Thederivatizing groups comprise typically 3 to 30 atoms in aliphatic oraromatic structures or can form terminal group spacers and link theglycomes to carriers such as solid phases or microparticels, polymericcarries such as oligosaccharides and/or polysaccharide, peptides,dendrimer, proteins, organic polymers such as plastics,polyethyleneglycol and derivatives, polyamines such as polylysines.

When the glycome comprises asparagine N-glycosides, A is preferably betaand R is linked asparagine or asparagine peptide. The peptide part maycomprise multiple different aminoacid residues and typically multipleforms of peptide with different sequences derived from natural proteinscarrying the N-glycans in cell materials according to the invention. Itis realized that for example proteolytic release of glycans may producemixture of glycopeptides. Preferably the peptide parts of theglycopeptides comprises mainly a low number of amino acid residues,preferably two to ten residues, more preferably two to seven amino acidresidues and even more preferably two to five aminoacid residues andmost preferably two to four amino acid residues when “mainly” indicatespreferably at least 60% of the peptide part, more preferably at least75% and most preferably at least 90% of the peptide part comprising thepeptide of desired low number of aminoacid residues.

The Preferred GN₂-N-glycan Core Structure(s)

The preferred GN₂-N-glycan core structure(s) and/or N-glycomes from stemcells according to the invention comprise structure(s) according to theformula NC2:

R₁Mβ4GNβ4(Fucα6)_(n)GNyR₂,

wherein n is 0 or 1 andwherein y is anomeric linkage structure α and/or β or linkage fromderivatized anomeric carbon andR₁ indicates 1-4, preferably 1-3, natural type carbohydrate substituentslinked to the core structures,R₂ is reducing end hydroxyl chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacid and/or peptides derived fromprotein.

The preferred compositions thus include one or several of the followingstructures

NC2a: Mα3{Mα6}Mβ4GNβ4{Fucα6}_(n1)GNyR₂NC2b: Mα6Mβ4GNβ4{Fucα6}_(n1)GNyR₂NC2c: Mα3Mβ4GNβ4{Fucα6}_(n1)GNyR₂

More preferably compositions comprise at least 3 of the structures ormost preferably both structures according to the formula NC2a and atleast both fucosylated and non-fucosylated with core structure(s) NC2band/or NC2c.

The Preferred GN₁—N-glycan Core Structure(s)

The preferred GN₁—N-glycan core structure(s) and/or N-glycomes from stemcells according to the invention comprise structure(s) according to theformula NC3:

R₁Mβ4GNyR₂,

wherein y is anomeric linkage structure α and/or β or linkage fromderivatized anomeric carbon andR₁ indicates 1-4, preferably 1-3, natural type carbohydrate substituentslinked to the core structures,R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagine N-glycoside aminoacids and/or peptides derived fromprotein.

Multi-mannose GN₁—N-glycan Core Structure(s)

The invention is specifically directed glycans and/or glycomes derivedfrom preferred cells according to the present invention when the naturalglycome or glycan comprises Multi-mannose GN₁—N-glycan core structure(s)structure(s) according to the formula NC4:

[R₁Mα3]_(n3){R₃Mα6}_(n2)Mβ4GNXyR₂,

R₁ and R3 indicate nothing or one or two, natural type carbohydratesubstituents linked to the core structures, when the substituents areα-linked mannose monosaccharide and/or oligosaccharides and the othervariables are as described above.

Furthermore common elongated GN₂—N-glycan core structures are preferredtypes of glycomes according to the invention

The preferred N-glycan core structures further include differentlyelongated GN₂—N-glycan core structures according to the formula NC5:

[R₁Mα3]_(n3){R₃Mα6}_(n2)Mβ4GNβ4{Fucα6}_(n1)GNyR₂,

wherein n1, n2 and n3 are either 0 or 1 andwherein y is anomeric linkage structure α and/or β or linkage fromderivatized anomeric carbon andR₁ and R₃ indicate nothing or 1-4, preferably 1-3, most preferably oneor two, natural type carbohydrate substituents linked to the corestructures,R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagine N-glycoside aminoacids and/or peptides derived fromprotein,GN is GlcNAc, M is mannosyl-, [ ]indicate groups either present orabsent in a linear sequence.{ } indicates branching which may be also present or absent.with the provision that at least n2 or n3 is 1. Preferably the inventionis directed to compositions comprising with all possible values of n2and n3 and all saccharide types when R1 and/or are R1 areoligosaccharide sequences or nothing.

Preferred N-glycan Types in Glycomes Comprising N-glycans

The present invention is preferably directed to N-glycan glycomescomprising one or several of the preferred N-glycan core types accordingto the invention. The present invention is specifically directed tospecific N-glycan core types when the compositions comprise N-glycan orN-glycans from one or several of the groups Low mannose glycans, Highmannose glycans, Hybrid glycans, and Complex glycans, in a preferredembodiment the glycome comrise substantial amounts of glycans from atleast three groups, more preferably from all four groups.

Major Subtypes of N-glycans in N-linked Glycomes

The invention revealed certain structural groups present in N-linkedglycomes. The grouping is based on structural features of glycan groupsobtained by classification based on the monosaccharide compositions andstructural analysis of the structurel groups. The glycans were analysedby NMR, specific binding reagents including lectins and antibodies andspecific glycosidases releasing monosaccharide residues from glycans.The glycomes are preferably analysed as neutral and acidic glycomes

The Major Neutral Glycan Types

The neutral glycomes mean glycomes comprising no acidic monosaccharideresidues such as sialic acids (especially NeuNAc and NeuGc), HexA(especially GlcA, glucuronic acid) and acid modification groups such asphosphate and/or sulphate esters. There are four major types of neutralN-linked glycomes which all share the common N-glycan core structure:High-mannose N-glycans, low-mannose N-glycans, hydrid type and complextype N-glycans. These have characteristic monosaccharide compositionsand specific substructures. The complex and hybrid type glycans mayinclude certain glycans comprising monoantennary glycans.

The groups of complex and hybrid type glycans can be further analysedwith regard to the presence of one or more fucose residues. Glycanscontaining at least one fucose units are classified as fucosylated.Glycans containing at least two fucose residues are considered asglycans with complex fucosylation indicating that other fucose linlages,in addition to the α1,6-linkage in the N-glycan core, are present in thestructure. Such linkages include α1,2-, α1,3-, and α1,4-linkage.

Furthermore the complex type N-glycans may be classified based on therelations of HexNAc (typically GlcNAc or GalNAc) and Hex residues(typically Man, Gal). Terminal HexNAc glycans comprise at least threeHexNAc units and at least two Hexose units so that the number of Hex Nacresidues is at least larger or equal to the number of hexose units, withthe provisiont that for non branched, monoantennary glycans the numberof HexNAcs is larger than number of hexoses.

This consideration is based on presence of two GlcNAc units in the coreof N-glycan and need of at least two Mannose units to for a singlecomplex type N-glycan branch and three mannose to form a trimannosylcore structure for most complex type structures. A specific group ofHexNAc N-Glycans contains the same number of HexNAcs and Hex units, whenthe number is at least 5.

Preferred Mannose Type Structures

The invention is for ther directed to glycans comprosing terminalMannose such as Mα6-residue or both Manα6- and Manα3-residues,respectively, can additionally substitute other Mα2/3/6 units to form aMannose-type structures including hydrid, low-Man and High-Manstructures according to the invention.

Preferred high- and low mannose type structures with GN2-core structureare according to the Formula M2:

[Mα2]_(n1)[Mα3]_(n2){[Mα2]_(n3)[Mα6)]_(n4)}[Mα6]_(n5){[Mα2]_(n6)[Mα2]_(n7)[Mα3]_(n8)}Mβ4GNβ4[{Fucα6}]_(m)GNyR₂

wherein p, n1, n2, n3, n4, n5, n6, n7, n8, and m are eitherindependently 0 or 1; with the proviso that when n2 is 0, also n1 is 0;when n4 is 0, also n3 is 0; when n5 is 0, also n1, n2, n3, and n4 are 0;when n7 is 0, also n6 is 0; when n8 is 0, also n6 and n7 are 0;y is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon, andR₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacid and/or peptides derived fromprotein;[ ] indicates determinant either being present or absent depending onthe value of n1, n2, n3, n4, n5, n6, n7, n8, and m; and{ } indicates a branch in the structure.

Preferred yR₂-structures include [β-N-Asn]_(p), wherein p is either 0 or1.

Preferred Mannose Type Glycomes Comprising GN1-core Structures

As described above a preferred variant of N-glycomes comprising onlysingle GlcNAc-residue in the core. Such structures are especiallypreferred as glycomes produced by endo-N-acetylglucosaminidase enzymesand Soluble glycomes. Preferred Mannose type glycomesnclude structuresaccording to the Formula M2

[Mα2]_(n1)[Mα3]_(n2){[Mα2]_(n3)[Mα6)]_(n4)}[Mα6]_(n5){[Mα2]_(n6)[Mα2]_(n7)[Mα3]_(n8)}Mβ4GNyR₂

Fucosylated high-mannose N-glycans according to the invention havemolecular compositions Man₅₋₉GlcNAc₂Fuc₁. For the fucosylatedhigh-mannose glycans according to the formula, the sum of n1, n2, n3,n4, n5, n6, n7, and n8 is an integer from 4 to 8 and m is 0.

The low-mannose structures have molecular compositionsMan₁₋₄GlcNAc₂Fuc₀₋₁. They consist of two subgroups based on the numberof Fuc residues: 1) nonfucosylated low-mannose structures have molecularcompositions Man₁₋₄GlcNAc₂ and 2) fucosylated low-mannose structureshave molecular compositions Man₁₋₄GlcNAc₂Fuc₁. For the low mannoseglycans the sum of n1, n2, n3, n4, n5, n6, n7, and n8 is less than orequal to (m+3); and preferably n1, n3, n6, and n7 are 0 when m is 0.

Low Mannose Glycans

The invention revealed a very unusual group of glycans in N-glycomes ofthe invention defined here as low mannose N-glycans. These are notclearly linked to regular biosynthesis of N-glycans, but may representunusual biosynthetic midproducts or degradation products. The lowmannose glycans are especially characteristics changing during thechanges of cell status, the differentiation and other changes accordingto the invention, for examples changes associated with differentiationstatus of embryonal-type stem cells and their differentiated productsand control cell materials. The invention is especially directed torecognizing low amounts of low-mannose type glycans in cell types, suchas stem cells, preferably embryonal type stem cells with low degree ofdifferentiation.

The invention revealed large differences between the low mannose glycanexpression in the early human blood cell glycomes, especially indifferent preferred cell populations from human cord blood.

The invention is especially directed to the use of specific low mannoseglycan comprising glycomes for analysis of early human blood glycomesespecially glycomes from cord blood.

The invention further revealed specific mannose directed recognitionmethods useful for recognizing the preferred glycomes according to theinvention. The invention is especially directed to combination ofglycome analysis and recognition by specific binding agents, mostpreferred binding agent include enzymes and theis derivatives. Theinvention further revealed that specific low mannose glycans of the lowmannose part of the glycomes can be recognized by degradation byspecific α-mannosidase (Man₂₋₄GlcNAc₂Fuc₀₋₁) or β-mannosidase(Man₁GlcNAc₂Fuc₀₋₁) enzymes and optionally further recognition of smalllow mannose structures, even more preferably low mannose structurescomprising terminal Manβ4-structures according to the invention.

The low mannose N-glycans, and preferred subgroups and individualstructures thereof, are especially preferred as markers of the novelglycome compositions of the cells according to the invention useful forcharacterization of the cell types.

The low-mannose type glycans includes a specific group of α3- and/orα6-linked mannose type structures according to the invention including apreferred terminal and core structure types according to the invention.

The inventions further revealed that low mannose N-glycans comprise aunique individual structural markers useful for characterization of thecells according to the invention by specific binding agents according tothe invention or by combinations of specific binding agents according tothe invention.

Neutral low-mannose type N-glycans comprise one to four or five terminalMan-residues, preferentially Manoa structures; for exampleManα₀₋₃Manβ4GlcNAcβ4GlcNAc(β-N-Asn) orManα₀₋₄Manβ4GlcNAcβ4(Fucα6)GlcNAc(β-N-Asn).

Low-mannose N-glycans are smaller and more rare than the commonhigh-mannose N-glycans (Man₅₋₉GlcNAc₂). The low-rnannose N-glycansdetected in cell samples fall into two subgroups: 1) non-fucosylated,with composition Man_(n)GlcNAc₂, where 1≦n≦4, and 2) core-fucosylated,with composition Man_(n)GlcNAc₂Fuc₁, where 1≦n≦5. The largest of thedetected low-mannose structure structures is Man₅GlcNAc₂Fuc₁ (m/z 1403for the sodium adduct ion), which due to biosynthetic reasons mostlikely includes the structure below (in the figure the glycan is freeoligosaccharide and β-anomer; in glycoproteins in tissues the glycan isN-glycan and β-anomer):

Preferred General Molecular Structural Features of Low Man Glycans

According to the present invention, low-mannose structures arepreferentially identified by mass spectrometry, preferentially based oncharacteristic Hex₁₋₄HexNAc₂dHex₀₋₁ monosaccharide composition. Thelow-mannose structures are further preferentially identified bysensitivity to exoglycosidase digestion, preferentially α-mannosidase(Hex₂₋₄HexNAc₂dHexc₀₋₁) or β-mannosidase (Hex₁HexNAc₂dHex₀₋₁) enzymes,and/or to endoglycosidase digestion, preferentially N-glycosidase Fdetachment from glycoproteins, Endoglycosidase H detachment fromglycoproteins (only Hex₁₋₄HexNAc₂ liberated as Hex₁₋₄HexNAc₁), and/orEndoglycosidase F2 digestion (only Hex₁₋₄HexNAc₂dHex₁ digested toHex₁₋₄HexNAc₁). The low-mannose structures are further preferentiallyidentified in NMR spectroscopy based on characteristic resonances of theManβ4GlcNAcβ4GlcNAc N-glycan core structure and Manα residues attachedto the Manβ4 residue.

Several preferred low Man glycans described above can be presented in asingle Formula:

[Mα3]_(n2){[Mα6)]_(n4)}[Mα6]_(n5){([Mα3]_(n8)}Mβ4GNβ4[{Fucα6}]_(m)GNyR₂

wherein p, n2, n4, n5, n8, and m are either independently 0 or 1; withthe proviso that when n2 is 0, also n1 is 0; when n4 is 0, also n3 is 0;when n5 is 0, also n1, n2, n3, and n4 are 0; when n7 is 0, also n6 is 0;when n8 is 0, also n6 and n7 are 0; the sum of n1, n2, n3, n4, n5, n6,n7, and n8 is less than or equal to (m+3); [ ] indicates determinanteither being present or absent depending on the value of n2, n4, n5, n8,and m; and{ } indicates a branch in the structure;y and R2 are as indicated above.

Preferred non-fucosylated low-mannose glycans are according to theformula:

[Mα3]_(n2)([Mα6)]_(n4))[Mα6]_(n5){[Mα3]_(n8)}Mβ4GNβ4GNyR₂

wherein p, n2, n4, n5, n8, and m are either independently 0 or 1, withthe provisio that when n5 is 0, also n2 and n4 are 0, and preferablyeither n2 or n4 is 0, [ ] indicates determinant either being present orabsentdepending on the value of, n2, n4, n5, n8,{ } and 0 indicates a branch in the structure,y and R2 are as indicated above.

Preferred Individual Structures of Non-fucosylated Low-mannose GlycansSpecial Small Structures

Small non-fucosylated low-mannose structures are especially unsual amongknown N-linked glycans and characteristic glycans group useful forseparation of cells according to the present invention. These include:

Mβ4GNβ4GNyR₂ Mα6Mβ4GNβ4GNyR₂ Mα3Mβ4GNβ4GNyR₂ and Mα6{Mα3}Mβ4GNβ4GNyR₂.

Mβ4GNβ4GNyR₂ trisaccharide epitope is a preferred common structure aloneand together with its mono-mannose derivatives Mα6Mβ4GNβ4GNyR₂ and/orMα3Mβ4GNβ4GNyR₂, because these are characteristic structures commonlypresent in glycomes according to the invention. The invention isspecifically directed to the glycomes comprising one or several of thesmall non-fucosylated low-mannose structures. The tetrasaccharides arein a specific embodiment preferred for specific recognition directed toα-linked, preferably α3/6-linked Mannoses as preferred terminalrecognition element.

Special Large Structures

The invention further revealed large non-fucosylated low-mannosestructures that are unsual among known N-linked glycans and have specialcharacteristic expression features among the preferred cells accordingto the invention. The preferred large structures include[Mα3]_(n2)([Mα6]_(n4))Mα6{Mα3}Mβ4GNβ4GNyR₂

more specifically

Mα6Mα6{Mα3}Mβ4GNβ4GNyR₂ Mα3Mα6{Mα3}Mβ4GNβ4GNyR₂ andMα3(Mα6)Mα6{Mα3}Mβ4GNβ4GNyR₂.

The hexasaccharide epitopes are preferred in a specific embodiment asrare and characteristic structures in preferred cell types and asstructures with preferred terminal epitopes. The heptasaccharide is alsopreferred as structure comprising a preferred unusual terminal epitopeMα3(Mα6)Mα useful for analysis of cells according to the invention.

Preferred fucosylated low-mannose glycans are derived according to theformula:

[Mα3]_(n2){[Mα6]_(n4)}[Mα6]_(n5){[Mα3]_(n8)}Mβ4GNβ4(Fucα6)GNyR₂

wherein p, n2, n4, n5, n8, and m are either independently 0 or 1, withthe provisio that when n5 is 0, also n2 and n4 are 0, [ ] indicatesdeterminant either being present or absent depending on the value of n1,n2, n3, n4, ( ) indicates a branch in the structure; and wherein n1, n2,n3, n4 and m are either independently 0 or 1,with the provisio that when n3 is 0, also n1 and n2 are 0,[ ] indicates determinant either being present or absentdepending on the value of n1, n2, n3, n4 and m,{ } and ( ) indicate a branch in the structure.

Preferred Individual Structures of Fucosylated Low-mannose Glycans

Small fucosylated low-mannose structures are especially unusual amongknown N-linked glycans and form a characteristic glycan group useful forseparation of cells according to the present invention. These include:

Mβ4GNβ(Fucα6)GNyR₂ Mα6Mβ4GNβ4(Fucα6)GNyR₂ Mα3Mβ4GNβ4(Fucα6)GNyR₂ andMα6{Mα3}Mβ4GNβ4(Fucα6)GNyR₂.

Mβ4GNβ4(Fucα6)GNyR₂ tetrasaccharide epitope is a preferred commonstructure alone and together with its mono-mannose derivativesMα6Mβ4GNβ4(Fucα6)GNyR₂ and/orMα3Mβ4GNβ4(Fucα6)GNyR₂, because these are commonly presentcharacteristics structures in glycomes according to the invention. Theinvention is specifically directed to the glycomes comprising one orseveral of the small non-fucosylated low-mannose structures. Thetetrasaccharides are in a specific embodiment preferred for specificrecognition directed to C-linked, preferably α3/6-linked Mannoses aspreferred terminal recognition element.

Special Large Structures

The invention further revealed large fucosylated low-mannose structuresare unsual among known N-linked glycans and have special characteristicexpression features among the preferred cells according to theinvention. The preferred large structure includes[Mα3]_(n2)([Mα6]_(n4))Mα6{Mα3}Mβ4GNβ4(Fucα6)GNyR₂

more specifically

Mα6Mα6{Mα3}Mβ4GNβ4(Fucα6)GNyR₂ Mα3Mα6{Mα3}Mβ4GNβ4(Fucα6)GNyR₂ andMα3(Mα6)Mα6{Mα3}Mβ4GNβ4(Fucα6)GNyR₂.

The heptasaccharide epitopes are preferred in a specific embodiment asrare and characteristic structures in preferred cell types and asstructures with preferred terminal epitopes. The octasaccharide is alsopreferred as structure comprising a preferred unusual terminal epitopeMα3(Mα6)Mα useful for analysis of cells according to the invention.

Preferred Non-reducing End Terminal Mannose-epitopes

The inventors revealed that mannose-structures can be labeled and/orotherwise specifically recognized on cell surfaces or cell derivedfractions/matrials of specific cell types. The present invention isdirected to the recognition of specific mannose epitopes on cellsurfaces by reagents binding to specific mannose structures from cellsurfaces.

The preferred reagents for recognition of any structures according tothe invention include specific antibodies and other carbohydraterecognizing binding molecules. It is known that antibodies can beproduced for the specific structures by various immunization and/orlibrary technologies such as phage display methods representing variabledomains of antibodies. Similarily with antibody library technologies,including aptamer technologies and including phage display for peptides,exist for synthesis of library molecules such as polyamide moleculesincluding peptides, especially cyclic peptides, or nucleotide typemolecules such as aptamer molecules.

The invention is specifically directed to specific recognitionhigh-mannose and low-mannose structures according to the invention. Theinvention is specifically directed to recognition of non-reducing endterminal Manα-epitopes, preferably at least disaccharide epitopes,according to the formula:

[Mα2]_(m1)[Mαx]_(m2)[Mα6]_(m3){{[Mα2]_(m9)[Mα2]_(m8)[Mα3]_(m7)}_(m10)(Mβ4[GN]_(m4))_(m5)}_(m6)yR₂

wherein m1, m2, m3, m4, m5, m6, m7, m8, m9 and m10 are independentlyeither 0 or 1; with the proviso that when m3 is 0, then m1 is 0 and,when m7 is 0 then either m1-5 are 0 and m8 and m9 are 1 formingMα2Mα2-disaccharide or both m8 and m9 are 0y is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon, andR₂ is reducing end hydroxyl, chemical reducing end derivativeand x is linkage position 3 or 6 or both 3 and 6 forming branchedstructure,{ } indicates a branch in the structure.

The invention is further directed to terminal Mα2-containing glycanscontaing at least one Mα2-group and preferably Mα2-group on each, branchso that m1 and at least one of m8 or m9 is 1. The invention is furtherdirected to terminal Mα3 and/or Mα6-epitopes without terminalMα2-groups, when all m1, m8 and m9 are 1.

The invention is further directed in a preferred embodiment to theterminal epitopes linked to a Mβ-residue and for application directed tolarger epitopes. The invention is especially directed toMβ4GN-comprising reducing end terminal epitopes.

The preferred terminal epitopes comprise typically 2-5 monosaccharideresidues in a linear chain. According to the invention short epitopescomprising at least 2 monosaccharide residues can be recognized undersuitable background conditions and the invention is specificallydirected to epitopes comprising 2 to 4 monosaccharide units and morepreferably 2-3 monosaccharide units, even more preferred epitopesinclude linear disaccharide units and/or branched trisaccharidenon-reducing residue with natural anomeric linkage structures atreducing end. The shorter epitopes may be preferred for specificapplications due to practical reasons including effective production ofcontrol molecules for potential binding reagents aimed for recognitionof the structures.

The shorter epitopes such as Mα2M-may is often more abundant on targetcell surface as it is present on multiple arms of several commonstructures according to the invention.

Preferred Disaccharide Epitopes Includes

Manα2Man, Manα3Man, Manα6Man, and more preferred anomeric formsManα2Manα, Manα3Manα, Manα6Manα, Manα3Manα and Manα6Manα.

Preferred branched trisaccharides includes Manα3(Manα6)Man,Manα3(Manα6)Manα, and Manα3(Manα6)Manα.

The invention is specifically directed to the specific recognition ofnon-reducing terminal Manα2-structures especially in context ofhigh-mannose structures.

The invention is specifically directed to following linear terminalmannose epitopes:

a) preferred terminal Manα2-epitopes including following oligosaccharidesequences:

Manα2Man, Manα2Manα, Manα2Manα2Man, Manα2Manα3Man, Manα2 Manα6Man,Manα2Manα2Manα, Manα2Manα3Manβ, Manα2Manα6Manα, Manα2Manα2Manα3Man,Manα2Manα3Manα6Man, Manα2Manα6Manα6Man Manα2Manα2Manα3Manβ,Manα2Manα3Manα6Manβ, Manα2Manα6Manα6Manβ;

The invention is further directed to recognition of and methods directedto non-reducing end terminal Manα3- and/or Manα6-comprising targetstructures, which are characteristic features of specifically importantlow-mannose glycans according to the invention. The preferred structuralgroups includes linear epitopes according to b) and branched epitopesaccording to the c3) especially depending on the status of the targetmatrial.

b) preferred terminal Manα3- and/or Manα6-epitopes including followingoligosaccharide sequences:Manα3Man, Manα6Man, Manα3Manβ, Manα6Manβ, Manα3Manα, Manα6Manα,Manα3Manα6Man, Manα6Manα6Man, Manα3Manα6Manβ, Manα6Manα6Manβ and tofollowingc) branched terminal mannose epitopes, are preferred as characteristicstructures of especially high-mannose structures (c1 and c2) andlow-mannose structures (c3), the preferred branched epitopes include:c1) branched terminal Manα2-epitopes

Manα2Manα3(Manα2Manα6)Man, Manα2Manα3(Manα2Manα6)Manα,Manα2Manα3(Manα2Manα6)Manα6Man, Manα2Manα3(Manα2Manα6)Manα6Manβ,Manα2Manα3(Manα2Manα6)Manα6(Manα2Manα3)Man,Manα2Manα3(Manα2Manα6)Manα6(Manα2Manα2Manα3)Man,Manα2Manα3(Manα2Manα6)Manα6(Manα2Manα3)ManβManα2Manα3(Manα2Manα6)Manα6(ManαManα2Manα3)Manβ

c2) branched terminal Manα2- and Manα3 or Manα6-epitopes according toformula when m1 and/or m8 and/m9 is 1 and the molecule comprise at leastone nonreducing end terminal Manα3 or Manα6-epitopec3) branched terminal Manα3 or Manα6-epitopes

Manα3(Manα6)Man, Manα3(Manα6)Manβ, Manα3(Manα6)Manα,Manα3(Manα6)Manα6Man, Manα3(Manα6)Manα6Manβ,Manα3(Manα6)Manα6(Manα3)Man, Manα3(Manα6)Manα6(Manα3)Manβ

The present invention is further directed to increase of selectivity andsensitivity in recognition of target glycans by combining recognitionmethods for terminal Manα2 and Manα3 and/or Manα6-comprising structures.Such methods would be especially useful in context of cell materialaccording to the invention comprising both high-mannose and low-mannoseglycans.

Complex Type N-glycans

According to the present invention, complex-type structures arepreferentially identified by mass spectrometry, preferentially based oncharacteristic monosaccharide compositions, wherein HexNAc≧4 and Hex≧3.In a more preferred embodiment of the present invention, 4≦HexNAc≦20 and3≦Hex≦21, and in an even more preferred embodiment of the presentinvention, 4≦HexNAc≦10 and 3≦Hex≦11. The complex-type structures arefurther preferentially identified by sensitivity to endoglycosidasedigestion, preferentially N-glycosidase F detachment from glycoproteins.The complex-type structures are further preferentially identified in NMRspectroscopy based on characteristic resonances of theManα3(Manα6)Manβ4GlcNAcβ4GlcNAc N-glycan core structure and GlcNAcresidues attached to the Manα3 and/or Manα6 residues.

Beside Mannose-type glycans the preferred N-linked glycomes includeGlcNAcβ2-type glycans including Complex type glycans comprising onlyGlcNAcβ2-branches and Hydrid type glycan comprising both Mannose-typebranch and GlcNAcβ2-branch.

GlcNAcβ2-type Glycans

The invention revealed GlcNAcβ2Man structures in the glycomes accordingto the invention. Preferably GlcNAcβ2Man-structures comprise one orseveral of GlcNAcβ2Manα-structures, more preferably GlcNAcβ2Manα3 orGlcNAcβ2Manα6-structure.

The Complex type glycans of the invention comprise preferably twoGlcNAcβ2Manα structures, which are preferably GlcNAcβ2Manα3 andGlcNAcβ2Manα6-. The Hybrid type glycans comprise preferablyGlcNAcβ2Manα3-structure.

The present invention is directed to at least one of naturaloligosaccharide sequence structures and structures truncated from thereducing end of the N-glycan according to the Formula GNβ2

[R₁GNβ2]_(n1)[Mα3]_(n2){[R₃]_(n3)[GNβ2]_(n4)Mα6}_(n5)MβGNXyR2,

with optionally one or two or three additional branches according toformula [R_(x)GNβz]_(nx) linked to Mα6-, Mα3-, or Mβ4 and R_(x) may bedifferent in each branchwherein n1, n2, n3, n4, n5 and nx, are either 0 or 1, independently,with the proviso that when n2 is 0 then n1 is 0 and when n3 is 1 or/andn4 is 1 then n5 is also 1, and at least n1 or n4 is 1, or n3 is 1,when n4 is 0 and n3 is 1 then R₃ is a mannose type substituent ornothing andwherein X is glycosidically linked disaccharide epitope β4(Fucα6)_(n)GN,wherein n is 0 or 1, or X is nothing andy is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon, andR₁, R_(x) and R₃ indicate independently one, two or three, naturalsubstituents linked to the core structure,R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacids and/or peptides derivedfrom protein.[ ] indicate groups either present or absent in a linear sequence. { }indicates branching which may be also present or absent.Elongation of GlcNAcβ2-type Structures, Complex/hydrid Type Structures

The substituents R₁, R_(x) and R₃ may form elongated structures. In theelongated structures R₁, and R_(x) represent substituents of GlcNAc (GN)and R₃ is either substituent of GlcNAc or when n4 is 0 and n3 is 1 thenR3 is a mannose type substituent linked to mannosea6-branch forming aHybrid type structure. The substituents of GN are monosaccharide Gal,GalNAc, or Fuc or and acidic residue such as sialic acid or sulfate orfosfate ester.

GlcNAc or GN may be elongated to N-acetyllactosaminyl also marked asGalβGN or di-N-acetyllactosdiaminyl GalNAcβGlcNAc preferablyGalNAcβ4GlcNAc. LNβ2M can be further elongated and/or branched with oneor several other monosaccharide residues such as by galactose, fucose,SA or LN-unit(s) which may be further substituted by SAα-strutures,and/or Mα6 residue and/or Mα3 residues can be further substituted one ortwo β6-, and/or β4-linked additional branches according to the formula,

and/or either of Mα6 residue or Mα3 residue may be absentand/or Mα6-residue can be additionally substitutes other Manα units toform a hybrid type structuresand/or Manβ4 can be further substituted by GNβ4,and/or SA may include natural substituents of sialic acid and/or it maybe substituted by other SA-residues preferably by α8- or α9-linkages.

The SAα-groups are linked to either 3- or 6-position of neighboring Galresidue or on 6-position of GlcNAc, preferably 3- or 6-position ofneighboring Gal residue. In separately preferred embodiments theinvention is directed structures comprising solely 3-linked SA or6-linked SA, or mixtures thereof.

Hybrid Type Structures

According to the present invention, hybrid-type or monoantennarystructures are preferentially identified by mass spectrometry,preferentially based on characteristic monosaccharide compositions,wherein HexNAc=3 and Hex≧2. In a more preferred embodiment of thepresent invention 2≦Hex≦11, and in an even more preferred embodiment ofthe present invention 2≦Hex≦9. The hybrid-type structures are furtherpreferentially identified by sensitivity to exoglycosidase digestion,preferentially α-mannosidase digestion when the structures containnon-reducing terminal α-mannose residues and Hex≧3, or even morepreferably when Hex≧4, and to endoglycosidase digestion, preferentiallyN-glycosidase F detachment from glycoproteins. The hybrid-typestructures are further preferentially identified in NMR spectroscopybased on characteristic resonances of theManα3(Manα6)Manβ4GlcNAcβ4GlcNAc N-glycan core structure, a GlcNAcβresidue attached to a Manα residue in the N-glycan core, and thepresence of characteristic resonances of non-reducing terminal α-mannoseresidue or residues.

The monoantennary structures are further preferentially identified byinsensitivity to α-mannosidase digestion and by sensitivity toendoglycosidase digestion, preferentially N-glycosidase F detachmentfrom glycoproteins. The monoantennary structures are furtherpreferentially identified in NMR spectroscopy based on characteristicresonances of the Manα3Manβ4GlcNAcβ4GlcNAc N-glycan core structure, aGlcNAcβ residue attached to a Manα residue in the N-glycan core, and theabsence of characteristic resonances of further non-reducing terminalα-mannose residues apart from those arising from a terminal α-mannoseresidue present in a ManαManβ sequence of the N-glycan core.

The present invention is directed to at least one of naturaloligosaccharide sequence structures and structures truncated from thereducing end of the N-glycan according to the Formula HY1

R₁GNβ2Mα3{[R₃]_(n3)Mα6}Mβ4GNXyR₂,

wherein n3, is either 0 or 1, independently,

AND

wherein X is glycosidically linked disaccharide epitope β4(Fucα6)_(n)GN,wherein n is 0 or 1, or X is nothing andy is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon, andR₁ indicate nothing or substituent or substituents linked to GlcNAc, R₃indicates nothing or Mannose-substituent(s) linked to mannose residue,so that each of R₁, and R₃ may correspond to one, two or three, morepreferably one or two, and most preferably at least one naturalsubstituents linked to the core structure,R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacids and/or peptides derivedfrom protein.[ ] indicate groups either present or absent in a linear sequence. { }indicates branching which may be also present or absent.

Preferred Hybrid Type Structures

The preferred hydrid type structures include one or two additionalmannose residues on the preferred core structure.

R₁GNβ2Mα3{[Mα3]_(m1)([Mα6])_(m2)Mα6}Mβ4GNXyR₂,  Formula HY2

wherein n3, is either 0 or 1, and m1 and m2 are either 0 or 1,independently,{ } and ( ) indicates branching which may be also present or absent,other variables are as described in Formula HY1.

Furthermore the invention is directed to structures comprisingadditional lactosamine type structures on GNβ2-branch. The preferredlactosamine type elongation structures includes N-acetyllactosamines andderivatives, galactose, GalNAc, GlcNAc, sialic acid and fucose.

Preferred structures according to the formula HY2 include:

Structures containing non-reducing end terminal GlcNAcAs a specific preferred group of glycans

GNβ2Mα3{Mα3Mα6}Mβ4GNXyR₂, GNβ2Mα3{Mα6Mα6}Mβ4GNXyR₂,GNβ2Mα3{Mα3Mα6}Mα6)Mβ4GNXyR₂,

and/or elongated variants thereof

R₁GNβ2Mα3{Mα3Mα6}Mβ4GNXyR₂, R₁GNβ2Mα3{Mα6Mα6}Mβ4GNXyR₂,R₁GNβ2Mα3{Mα3(Mα6)Mα6}Mβ4GNXyR₂,

[R₁Gal[NAc]_(o2)βz]_(o1)GNβ2Mα3{[Mα3]_(m1)[(Mα6)]_(m2)Mα6}_(n5)Mβ4GNXyR₂,  FormulaHY3

wherein n1, n2, n3, n5, m1, m2, 01 and o2 are either 0 or 1,independently,z is linkage position to GN being 3 or 4 in a preferred embodiment 4,R₁ indicates on or two a N-acetyllactosamine type elongation groups ornothing,{ } and ( ) indicates branching which may be also present or absent,other variables are as described in Formula HY1.

Preferred structures according to the formula HY3 include especially

structures containing non-reducing end terminal Galβ, preferably Galβ3/4forming a terminal N-acetyllactosamine structure. These are preferred asa special group of Hybrid type structures, preferred as a group ofspecific value in characterization of balance of Complex N-glycanglycome and High mannose glycome:GalβzGNβ2Mα3{Mα3Mα6}Mβ4GNXyR₂, GalβzGNβ2Mα3{Mα6Mα6}Mβ4GNXyR₂,GalβzGNβ2Mα3{Mα3(Mα6)Mα6}Mβ4GNXyR₂,and/or elongated variants thereof preferred for carrying additionalcharacteristic terminal structures useful for characterization of glycanmaterialsR₁GalβzGNβ2Mα3{Mα3Mα6}Mβ4GNXyR₂,R₁GalβzGNβ2Mα3{Mα6Mα6}Mβ4GNXyR₂,R₁GalβzGNβ2Mα3{Mα3(Mα6)Mα6}Mβ4GNXyR₂. Preferred elongated materialsinclude structures wherein R₁ is a sialic acid, more preferably NeuNAcor NeuGc.

Complex N-glycan Structures

The present invention is directed to at least one of naturaloligosaccharide sequence structures and structures truncated from thereducing end of the N-glycan according to the Formula CO1

[R₁GNβ2]_(n1)[Mα3]_(n2){[R₃GNβ2]_(n4)Mα6}_(n5)Mβ4GNXyR₂

with optionally one or two or three additional branches according toformula [R_(x)GNβz]_(nx) linked to Mα6-, Mα3-, or Mβ4 and R_(x) may bedifferent in each branchwherein n1, n2, n4, n5 and nx, are either 0 or 1, independently,with the proviso that when n2 is 0 then n1 is 0 and when n4 is 1 then n5is also 1, and at least n1 is 1 or n4 is 1, and at least either of n1and n4 is 1andwherein X is glycosidically linked disaccharide epitope β4(Fucα6)_(n)GN,wherein n is 0 or 1, or X is nothing andy is anomeric linkage structure α and/or β or linkage from derivatizedanomeric carbon, and R₁, R_(x) and R₃ indicate independently one, two orthree, natural substituents linked to the core structure,R₂ is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacids and/or peptides derivedfrom protein.[ ] indicate groups either present or absent in a linear sequence. { }indicates branching which may be also present or absent.

Preferred Complex Type Structures Incomplete Monoantennary N-glycans

The present invention revealed incomplete Complex monoantennaryN-glycans, which are unusual and useful for characterization of glycomesaccording to the invention. The most of the in complete monoantennarystructures indicate potential degradation of biantennary N-glycanstructures and are thus preferred as indicators of cellular status. Theincomplete Complex type monoantennary glycans comprise only oneGNβ2-structure.

The invention is specifically directed to structures are according tothe Formula CO1 above when only n1 is 1 or n4 is one and mixtures ofsuch structures.

The preferred mixtures comprise at least one monoantennary complex typeglycans

A) with single branches from a likely degradative biosynthetic process:

R₁GNβ2Mα3β4GNXyR₂ R₃GNβ2Mα6Mβ4GNXyR₂ and

B) with two branches comprising mannose branches

B1) R₁GNβ2Mα3{Mα6}_(n5)Mβ4GNXyR₂

B2) Mα3{R₃GNβ2Mα6}_(n5)Mβ4GNXyR₂

The structure B2 is preferred with A structures as product ofdegradative biosynthesis, it is especially preferred in context of lowerdegradation of Manα3-structures. The structure B1 is useful forindication of either degradative biosynthesis or delay of biosyntheticprocess

Biantennary and Multiantennary Structures

The inventors revealed a major group of biantennary and multiantennaryN-glycans from cells according to the invention, the preferredbiantennary and multiantennary structures comprise two GNβ2 structures.

These are preferred as an additional characteristics group of glycomesaccording to the invention and are represented according to the FormulaCO2:

R₁GNβ2Mα3{R₃GNβ2Mα6}MβGNXyR₂

with optionally one or two or three additional branches according toformula [R_(x)GNβz]_(nx) linked to Mα6-, Mα3-, or Mβ4 and R_(x) may bedifferent in each branchwherein nx is either 0 or 1,and other variables are according to the Formula CO1.

Preferred Biantennary Structure

A biantennary structure comprising two terminal GNβ-epitopes ispreferred as a potential indicator of degradative biosynthesis and/ordelay of biosynthetic process. The more preferred structures areaccording to the Formula CO2 when R₁ and R₃ are nothing.

Elongated Structures

The invention revealed specific elongated complex type glycanscomprising Gal and/or GalNAc-structures and elongated variants thereof.Such structures are especially preferred as informative structuresbecause the terminal epitopes include multiple informative modificationsof lactosamine type, which characterize cell types according to theinvention. The present invention is directed to at least one of naturaloligosaccharide sequence structure or group of structures andcorresponding structure(s) truncated from the reducing end of theN-glycan according to the Formula CO3

[R₁Gal[NAc]_(o2)βz2]_(o1)GNβ2Mα3{[R₁Gal[NAc]_(o4)βz2]_(o3)GNβ2Mα6}Mβ4GNXyR₂,

with optionally one or two or three additional branches according toformula [R_(x)GNβz1]_(nx) linked to Mα6-, Mα3-, or Mβ4 and R_(x) may bedifferent in each branchwherein nx, o1, o2, o3, and o4 are either 0 or 1, independently,with the provisio that at least o1 or o3 is 1, in a preferred embodimentboth are 1z2 is linkage position to GN being 3 or 4, in a preferred embodiment 4,z1 is linkage position of the additional branches.R₁, Rx and R₃ indicate on or two a N-acetyllactosamine type elongationgroups or nothing,{ } and ( ) indicates branching which may be also present or absentother variables are as described in Formula CO1.

Galactosylated Structures

The inventors characterized especially directed to digalactosylatedstructure GalβzGNβ2Mα3{GalβzGNβ2Mα6}Mβ4GNXyR₂,

and monogalactosylated structures:GalβzGNβ2Mα3{GNβ2Mα6}Mβ4GNXyR₂,GNβ2Mα3{GalβzGNβ2Mα6}Mβ4GNXyR₂,and/or elongated variants thereof preferred for carrying additionalcharacteristic terminal structures useful for characterization of glycanmaterialsR₁GalβzGNβ2Mα3{R₃GalβzGNβ2Mα6}Mβ4GNXyR₂R₁GalβzGNβ2Mα3{GNβ2Mα6}Mβ4GNXyR₂, andGNβ2Mα3{R₃GalβzGNβ2Mα6}Mβ4GNXyR₂.

Preferred elongated materials include structures wherein R₁ is a sialicacid, more preferably NeuNAc or NeuGc.

LacdiNAc-structure Comprising N-glycans

The present invention revealed for the first time LacdiNAc,GalNacbGlcNAc structures from the cell according to the invention.Preferred N-glycan lacdiNAc structures are included in structuresaccording to the Formula CO1, when at least one the variable o2 and o4is 1.

The Major Acidic Glycan Types

The acidic glycomes mean glycomes comprising at least one acidicmonosaccharide residue such as sialic acids (especially NeuNAc andNeuGc) forming sialylated glycome, HexA (especially GlcA, glucuronicacid) and/or acid modification groups such as phosphate and/or sulphateesters.

According to the present invention, presence of phosphate and/orsulphate ester (SP) groups in acidic glycan structures is preferentiallyindicated by characteristic monosaccharide compositions containing oneor more SP groups. The preferred compositions containing SP groupsinclude those formed by adding one or more SP groups into non-SP groupcontaining glycan compositions, while the most preferential compositionscontaining SP groups according to the present invention are selectedfrom the compositions described in the acidic N-glycan fraction glycangroup tables. The presence of phosphate and/or sulphate ester groups inacidic glycan structures is preferentially further indicated by thecharacteristic fragments observed in fragmentation mass spectrometrycorresponding to loss of one or more SP groups, the insensitivity of theglycans carrying SP groups to sialidase digestion. The presence ofphosphate and/or sulphate ester groups in acidic glycan structures ispreferentially also indicated in positive ion mode mass spectrometry bythe tendency of such glycans to form salts such as sodium salts asdescribed in the Examples of the present invention. Sulphate andphosphate ester groups are further preferentially identified based ontheir sensitivity to specific sulphatase and phosphatase enzymetreatments, respectively, and/or specific complexes they form withcationic probes in analytical techniques such as mass spectrometry.

Complex N-glycan Glycomes, Sialylated

The present invention is directed to at least one of naturaloligosaccharide sequence structures and structures truncated from thereducing end of the N-glycan according to the Formula

[{SAα3/6}_(s1)LNβ2]_(r1)Mα3{({SAα3/6}_(s2)LNβ2)_(r2)Mα6}_(r8){M[β4GN[β4{Fucα6}_(r3)GN]_(r4)]_(r5)}_(r6)  (I)

with optionally one or two or three additional branches according toformula

{SAα3/6}_(s3)LNβ,  (IIb)

wherein r1, r2, r3, r4, r5, r6, r7 and r8 are either 0 or 1,independently, wherein s1, s2 and s3 are either 0 or 1, independently,with the proviso that at least r1 is 1 or r2 is 1, and at least one ofs1, s2 or s3 is 1.

LN is N-acetyllactosaminyl also marked as GalβGN ordi-N-acetyllactosdiaminyl GalNAcβGlcNAc preferably GalNAcβ4GlcNAc, GN isGlcNAc, M is mannosyl-, with the proviso LNβ2M or GNβ2M can be furtherelongated and/or branched with one or several other monosaccharideresidues such as by galactose, fucose, SA or LN-unit(s) which may befurther substituted by SAα-strutures,

and/or one LNβ can be truncated to GNβand/or Mα6 residue and/or Mα3 residues can be further substituted one ortwo β6-, and/or β4-linked additional branches according to the formula,and/or either of Mα6 residue or Mα3 residue may be absentand/or Mα6-residue can be additionally substitutes other Manα units toform a hybrid type structuresand/or Manβ4 can be further substituted by GNβ4,and/or SA may include natural substituents of sialic acid and/or it maybe substituted by other SA-residues preferably by α8- or α9-linkages.( ), { }, [ ] and [ ] indicate groups either present or absent in alinear sequence. { } indicates branching which may be also present orabsent.

The SAα-groups are linked to either 3- or 6-position of neighboring Galresidue or on 6-position of GlcNAc, preferably 3- or 6-position ofneighboring Gal residue. In separately preferred embodiments theinvention is directed structures comprising solely 3-linked SA or6-linked SA, or mixtures thereof. In a preferred embodiment theinvention is directed to glycans wherein r6 is 1 and r5 is 0,corresponding to N-glycans lacking the reducing end GlcNAc structure.

The LN unit with its various substituents can in a preferred generalembodiment represented by the formula:

[Gal(NAc)_(n1)α3]_(n2){Fucα2}_(n3)Gal(NAc)_(n4)β3/4{Fucα4/3}_(n5)GlcNAcβ

wherein n1, n2, n3, n4, and n5 are independently either 1 or 0,with the provisio thatthe substituents defined by n2 and n3 are alternative to presence of SAat the non-reducing end terminalthe reducing end GlcNAc-unit can be further β3- and/or β6-linked toanother similar LN-structure forming a poly-N-acetyllactosaminestructurewith the provision that for this LN-unit n2, n3 and n4 are 0,the Gal(NAc)β and GlcNAcβ units can be ester linked a sulphate estergroup,( ), and [ ] indicate groups either present or absent in a linearsequence; { } indicates branching which may be also present or absentLN unit is preferably Galβ4GN and/or Galβ3GN. The inventors revealedthat early human cells can express both types of N-acetyllactosamine,the invention is especially directed to mixtures of both structures.Furthermore the invention is directed to special relatively rear type 1N-acetyllactosamines, Galβ3GN, without any non-reducing end/sitemodification, also called lewis c-structures, and substitutedderivatives thereof, as novel markers of early human cells.

Occurrence of Structure Groups in Preferred Cell Types

In the present invention, glycan signals with preferentialmonosaccharide compositions can be grouped into structure groups basedon classification rules described in the present invention. The presentinvention includes parallel and overlapping classification systems thatare used for the classification of the glycan structure groups.

Glycan signals isolated from the N-glycan fractions from the cell typesstudied in the present invention are grouped into glycan structuregroups based on their preferential monosaccharide compositions accordingto the invention, in Table 46 for neutral N-glycan fractions and Table47 for acidic N-glycan fractions. Taken together, the analyses revealedthat all the structure groups according to the invention are present inthe studied cell types.

The invention is specifically directed to terminal HexNAc groups and/orother structure groups and/or combinations thereof as shown in theExamples describing and analysis of stem cell including hESC glycanstructure classification. Non-reducing terminal HexNAc residues could beliberated from the cell types studied in the present invention byspecific combinations of β-hexosaminidase and β-glucosaminidasedigestions, confirming the structural group classification of thepresent invention, and identifying terminal HexNAc residues as β-GlcNAcand/or β-GalNAc residues in the studied cell types. According to thepresent invention, specifically in hESC and cells differentiatedtherefrom the terminal HexNAc residues preferentially include bothβ-GlcNAc and β-GalNAc residues, more preferentially terminal β-GlcNAclinkages including bisecting GlcNAc linkages and other hybrid-type andcomplex-type GlcNAc linkages according to the present invention, andterminal GalNAc linkages including β4-linked GalNAc and mostpreferentially GalNAcβ4GlcNAcβ (LacdiNAc) structures according to thepresent invention.

Integrated Glycome Analysis Technology

The invention is directed to analysis of present cell materials based onsingle or several glycans (glycome profile) of cell materials accordingto the invention. The analysis of multiple glycans is preferablyperformed by physical analysis methods such as mass spectrometry and/orNMR.

The invention is specifically directed to integrated analysis processfor glycomes, such as total glycomes and cell surface glycomes. Theintegrated process represent various novel aspects in each part of theprocess. The methods are especially directed to analysis of low amountsof cells. The integrated analysis process includes

A) preferred preparation of substrate cell materials for analysis,including one or several of the methods: use of a chemical buffersolution, use of detergents, chemical reagents and/or enzymes.B) release of glycome(s), including various subglycome type based onglycan core, charge and other structural features, use of controlledreagents in the processC) purification of glycomes and various subglycomes from complexmixturesD) preferred glycome analysis, including profiling methods such as massspectrometry and/or NMR spectroscopyE) data processing and analysis, especially comparative methods betweendifferent sample types and quantitative analysis of the glycome data.

A. Preparation of Cell Materials

Cell substrate material and its preparation for total and cell surfaceglycome analysis. The integrated glycome analysis includes preferably acell preparation step to increase the availability of cell surfaceglycans. The cell preparation step preferably degrades either total cellmaterials or cell surface to yield a glycome for more effective glycanrelease. The degradation step preferably includes methods of physicaldegradation and/or chemical degradation. In a preferred embodiment atleast one physical and one chemical degradation methods are combined,more preferably at least one physical method is combined with at leasttwo chemical methods, even more preferably with at least three chemicalmethods.

The physical degration include degration by energy including thermaland/or mechanical energy directed to the cells to degrade cellstructures such as heating, freezing, sonication, and pressure. Thechemical degradation include use of chemicals and specificconcentrations of chemicals for distruption distruption of cellspreferably detergents including ionic and neutral detergents, chaotropicsalts, denaturing chemicals such as urea, and non-physiological saltconcentrations for distruption of the cells.

The glycome analysis according to the invention is divided to twomethods including Total cell glycomes, and Cell surface glycomes. Theproduction of Total cell glycomes involves degradation of cells byphysical and/or chemical degradation methods, preferably at least bychemical methods, more preferably by physical and chemical methods. TheCell surface glycomes is preferably released from cell surfacepreserving cell membranes intact or as intact as possible, such methodsinvolve preferably at least one chemical method, preferably enzymaticmethod. The cell surface glycomes may be alternatively released fromisolated cell membranes, this method involves typically chemical and/orphysical methods similarily as production of total cell glycomes,preferably at least use of detergents.

a. Total Cell Glycomes

The present invention revealed special methods for effectivepurification of released glycans from total cell derived materials sothat free oligosaccharides can be obtained. In a preferred embodiment atotal glycome is produced from a cell sample, which is degraded to formmore available for release of glycans. A preferred degraded form ofcells is detergent lysed cells optionally involving physical distruptionof cell materials.

Preferred detergents and reaction conditions include,

a1) ionic detergents, preferably SDS type anionic detergent comprisingan anionic group such as sulfate and an alkyl chain of 8-16 carbonatoms, more preferably the anionic detergent comprise 10-14 carbon atomsand it is most preferably sodium dodecyl sulfate (SDS), and/ora2) non-ionic detergents such as alkylglycosides comprising a hexose and4-12 carbon alkyl chain more preferably the alkyl chain comprises ahexoses being galactose, glucose, and/or mannose, more preferablyglucose and/or mannose and the alkyl comprises 6-10 carbon atoms,preferably the non-ionic detergent is octylglucoside

It is realized that various detergent combinations may be produced andoptimized. The combined use of an ionic, preferably anionic, andnon-ionic detergents according to the invention is especially preferred.

Preferred Cell Preparation Methods for Production of Total Cell Glycome

The preferred methods of cell degration for Total cell glycomes includephysical degration including at least heat treatment heat and chemicaldegration by a detergent method or by a non-detergent method preferablyenzymatic degradation, preferably heat treatment. Preferably twophysical degradation methods are included.

A preferred Non-detergent Method Includes

A non-detergent method is preferred for avoiding detergent in laterpurification. The preferred non-detergent method involves physicaldegradation of cells preferably pressure and or by heat and a chemicaldegradation by protease. A preferred non-detergent method includes:

i) cell degradation by physical methods, for example by pressure methodssuch as by French press.

The treatment is preferably performed quickly in cold temperatures,preferably at 0-2 degrees of Celsius, and more preferably at about 0 or1 degree of celsius and/or in the presence of glycosidase inhibitors.

ii) The degraded cells are further treated with chemical degradation,preferably by effective general protease, more preferably trypsin isused for the treatment. Preferred trypsin preparation according to theinvention does not cause glycan contamination to the sample/does notcontain glycans releasable under the reaction conditions.iii) optionally the physical degradation and chemical degradation arerepeated.iv) At the end of protease treatment the sample is boiled for furtherdenaturing the sample and the protease. The boling is performed attemperature denaturing/degrading further the sample and the proteaseactivity (conditions thus depend on the protease used) preferably about100 degrees Celsius for time enough for denaturing protease activitypreferably about 10-20 minutes for trypsin, more preferably about 15minutes.

Preferred Detergent Method for Production of Total Glycomes

The invention is in another preferred embodiment directed to detergentbased method for lysing cells. The invention includes effective methodsfor removal of detergents in later purification steps. The detergentmethods are especially preferred for denaturing proteins, which may bindor degrade glycans, and for degrading cell membranes to increase theaccessibility of intracellular glycans.

For the detergent method the cell sample is preferably a cell pelletproduced at cold tenperature by centrifuging cells but avoidingdistruption of the cells, optionally stored frozed and melted on ice.Optionally glycosidase inhibitors are used during the process.

The method includes following steps:

i) production of cell pellet preferably by centrifugation,ii) lysis by detergent on ice, the detergent is preferably an anionicdetergent according to the invention, more preferably SDS. Theconcentration of the detergent is preferably between about 0.1% and 5%,more preferably between 0.5%-3%, even more preferably between 0.5-1.5%and most preferably about 1% and the detergent is SDS (or between0.9-1.1%).the solution is preferably produced in ultrapure water,iii) mixing by effective degradation of cells, preferably mixing by aVortex-mixer as physical degradation step,iv) boiling on water bath, preferebly for 3-10 min, most preferablyabout 5 min (4-6 min) as second physical degradation step, it isrealized that even longer boiling may be performed for example up to 30min or 15 min, but it is not optimal because of evaporation samplev) adding one volume of non-ionic detergent, preferably alkyl-glycosidedetergent according to the invention, most preferablyn-octyl-β-D-glucoside, the preferred amount of the detergent is about5-15% as water solution, preferably about 10% of octyl-glucoside. Thenon-ionic detergent is especially preferred in case an enzyme sensitiveto SDS, such as a N-glycosidase, is to be used in the next reactionstep.andvi) incubation at room temperature for about 5 min to about 1-4 hours,more preferably less than half an hour, and most preferably about 15min.

Preferred Amount of Detergents in the Detergent Method

Preferably the anionic detergent and cationic detergent solutions areused in equal volumes. Preferably the solutions are about 1% SDS andabout 10% octyl-glucoside. The preferred amounts of the solutions arepreferably from 0.1 μl to about 2 μl, more preferably 0.15 μl to about1.5 μl per and most preferably from 0.16 μl to 1 μl per 100 000 cells ofeach solution. Lower amounts of the detergents are preferred if possiblefor reduction of the amount of detergent in later purification, highestamounts in relation to the cell amounts are used for practical reasonswith lowest volumes. It is further realized that corresponding weightamounts of the detergents may be used in volumes of about 10% to about1000%, or from about 20% to about 500% and even more effectively involumes from 30% to about 300% and most preferably in volumes of rangefrom 50% to about 150% of that described. It is realized that criticalmicellar concentration based effects may reduce the effect of detergentsat lowest concentrations.

In a preferred embodiment a practical methods using tip columns asdescribed in the invention uses about 1-3 μl of each detergent solution,more preferably 1.5-2.5 μl, and most preferably about 2 μl of thepreferred detergent solutions or corresponding detergent amounts areused for about 200 000 or less cells (preferably between 2000 and about250 000 cells, more preferably from 50 000 to about 250 000 cells andmost preferably from 100 000 to about 200 000 cells). Another practicalmethod uses uses about 2-10 μl of each detergent solution, morepreferably 4-8 μl, and most preferably about 5 μl (preferably between 4and 6 μl and more preferably between 4.5 and 5.5 μl) of detergentsolutions or corresponding amount of the detergents for lysis of cell ofa cell amount from about 200 000-3 million cells (preferred more exactranges include 200 000-3.5 million, 200 000 to 3 million and 200 000 to2.5 million cells), preferably a fixed amount (specific amount ofmicroliters preferably with the accuracy of at least 0.1 microliter) ina preferred range such as of 5.0 μl is used for the wider range of cells200 000-3 million. It was invented that is possible to handle similaritywider range of materials. It is further realized that the method can beoptimized so that exact amount of detergent, preferably within theranges described, is used for exact amount of cells, such method ispreferably an automized when there is possible variation in amounts ofsample cells.

b. Cell Surface Glycomes

In another preferred embodiment the invention is directed to release ofglycans from intact cells and analysis of released cell surfaceglycomes. The present invention is directed to specific buffer andenzymatic cell pre-modification conditions that would allow theefficient use of enzymes for release and optionally modification andrelease of glycans.

B. The Glycan Release Methods

The invention is directed to various enzymatic and chemical methods torelease glycomes. The release step is not needed for soluble glycomesaccording to the invention. The invention further revealed solubleglycome components which can be isolated from the cells using methodsaccording to the invention.

C. Purification of glycans from cell derived materials The purificationof glycome materials form cell derived molecules is a difficult task. Itis especially difficult to purify glycomes to obtain picomol or lownanomol samples for glycome profiling by mass spectrometry orNMR-spectrometry. The invention is especially directed to production ofmaterial allowing quantitative analysis over a wide mass range. Theinvention is specifically directed to the purification ofnon-derivatized or reducing end derivatized glycomes according to theinvention and glycomes containing specific structural characteristicsaccording to the invention. The structural characteristics wereevaluated by the preferred methods according to the invention to producereproducible and quantitative purified glycomes.

Glycan Purification Process Steps

The glycan purification method according to the present inventionconsists of at least one of purification options, preferably in specificcombinations described below, including one or several of following thefollowing purification process steps in varying order:

6) Precipitation-extraction; 7) Ion-exchange;

8) Hydrophobic interaction;9) Hydrophilic interaction; and10) Affinity to carbon materials especially graphitized carbon.

Preurification and Purification Process Steps

In general the purification steps may be divided to two majorcategories:

Prepurification steps to remove major contaminations and purificationsteps usually directed to specific binding and optionally fiactionationog glycomes

a) Prepurification to Remove Non-carbohydrate Impurities

The need for prepurification depends on the type and amounts of thesamples and the amounts of impurities present. Certain samples it ispossible to omit all or part of the prepurification steps. Theprepurification steps are aimed for removal of major non-carbohydrateimpurities by separating the impurity and the glycome fraction(s) to bepurified to different phases by precipitation/extraction or binding tochromatography matrix and the separating the impurities from the glycomefraction(s).

The prepurification steps include one, two or three of following majorsteps: Precipitation-extraction, Ion-exchange, Hydrophobic interaction.

The precipitation and/or extraction is based on the high hydrophilicnature of glycome compositions and components, which is useful forseparation from different cellular components and chernicals. Theprepurification ion exchange chromatography is directed to removal ofclasses molecules with different charge than the preferred glycome orglycome fraction to be studied. This includes removal of salt ions andaminoacids, and peptides etc. The glycome may comprise only negativecharges or in more rare case also only positive charges and the samecharge is selected for the chromatography matrix for removal of theimpurities for the same charge without binding the glycome atprepurification.

In a preferred embodiment the invention is directed to removal ofcationic impurities from glycomes glycomes containing neutral and/ornegatively charged glycans. The invention is further directed to useboth anion and cation exchange for removal of charged impurities fromnon-charged glycomes. The preferred ion exchange and cation exhangematerials includes polystyrene resins such as Dowex resins.

The hydrophilic chromatography is preferably aimed for removal ofhydrophobic materials such as lipids detergents and hydrophobic proteinmaterials.

It is realized that different combinations of the prepurification areusuful depending on the cell preparation and sample type. Preferredcombinations of the prepurification steps include:Precipitation-extraction and Ion-exchange; Precipitation-extraction andHydrophobic interaction; and Ion-exchange and Hydrophobic interaction.The two prepurification steps are preferably performed in the givenorder.

Purification Steps Including Binding and Optionally Fractionation ofGlycomes

The purification steps utilize two major concepts for binding tocarbohydrates and combinations thereof: a) Hydrophilic interactions andb) Ion exhange

a) Hydrophilic Interactions

The present invention is specifically directed to use of matrices withrepeating polar groups with affinity for carbohydrates for purificationof glycome materials according to the invention in processes accordingot the invention. The hydrophilic interaction material may includeadditional ion exchange properties.

The preferred hydrophilic interaction materials includes carbohydratematerials such as carbohydrate polymers in presence of non-polar organicsolvents. A especially preferred hydrophilic interaction chromatographymatrix is cellulose.

A specific hydrophilic interaction material includes graphitized carbon.The graphitized carbon separates non-charged carbohydrate materialsbased mainly on the size on the glycan. There are also possible ionexchange effects. In a preferred embodiment the invention is directed tographitized carbon chromatography of prepurified samples after desaltingand removal of detergents.

The invention is specifically directed to purification ofnon-derivatized glycomes and neutral glycomes by cellulosechromatography. The invention is further directed to purification ofnon-derivatized glycomes and neutral glycomes by graphitized carbonchromatography. In a preferred embodiment the purification according tothe invention includes both cellulose and graphitized carbonchromatography.

b) Ion Exchange

The glycome may comprise only negative charges or in more rare case alsoonly positive charges. At purification stage the ion exchange materialis selected to contain opposite charge than the glycome or glycomefraction for binding the glycome. The invention is especially directedto the use of anion exchange materials for binding of negatively chargedPreferred ion exchange materials includes ion exchange and especiallyanion exhange materials includes polystyrene resins such asDowex-resins, preferably quaternary amine resins anion exchange orsulfonic acid cation exchange resins

It was further revealed that even graphitized carbon can be used forbinding of negatively charged glycomes and the materials can be elutedfrom the carbon separately from the neutral glycomes or glycomefractions according to the invention.

The invention is specifically directed to purification of anionicglycomes by anion exchange chromatography.

The invention is specifically directed to purification of anionicglycomes by anion exchange chromatography.

The invention is further directed to purification of anionic glycomes bycellulose chromatography. The preferred anionic glycomes comprise sialicacid and/or sulfo/fosfo esters, more preferably both sialic acid andsulfo/fosfo esters. A preferred class of sulfo/fosfoester glycomes arecomplex type N-glycans comprising sulfate esters.

Prepurification and Purification Steps in Detail

1) Precipitation-extraction may include precipitation of glycans orprecipitation of contaminants away from the glycans. Preferredprecipitation methods include:

1. Glycan material precipitation, for example acetone precipitation ofglycoproteins, oligosaccharides, glycopeptides, and glycans in aqueousacetone, preferentially ice-cold over 80% (v/v) aqueous acetone;optionally combined with extraction of glycans from the precipitate,and/or extraction of contaminating materials from the precipitate;2. Protein precipitation, for example by organic solvents ortrichloroacetic acid, optionally combined with extraction of glycansfrom the precipitate, and/or extraction of contaminating materials fromthe precipitate;3. Precipitation of contaminating materials, for example precipitationwith trichloroacetic acid or organic solvents such as aqueous methanol,preferentially about 2/3 aqueous methanol for selective precipitation ofproteins and other non-soluble materials while leaving glycans insolution;

2) Ion-exchange may include ion-exchange purification or enrichment ofglycans or removal of contaminants away from the glycans. Preferredion-exchange methods include:

1. Cation exchange, preferably for removal of contaminants such assalts, polypeptides, or other cationizable molecules from the glycans;and2. Anion exchange, preferably either for enrichment of acidic glycanssuch as sialylated glycans or removal of charged contaminants fromneutral glycans, and also preferably for separation of acidic andneutral glycans into different fractions.

3) Hydrophilic interaction may include purification or enrichment ofglycans due to their hydrophilicity or specific adsorption tohydrophilic materials, or removal of contaminants such as salts awayfrom the glycans. Preferred hydrophilic interaction methods include:

1. Hydrophilic interaction chromatography with specific organic orinorganic polar interaction materials, preferably for purification orenrichment of glycans and/or glycopeptides;2. Preferably adsorption of glycans to carbohydrate materials,preferably to cellulose in hydrophobic solvents for their purificationor enrichment, preferably to microcrystalline cellulose, and elution bypolar solvents such as water and or alchol, which is preferably ethanolor methanol. The solvent system for absorption comprise preferably

-   -   i) a hydrophobic alcohol comprising about three to five carbon        atoms, including propanols, butanols, and pentanols, more        preferably being n-butanol;    -   ii) a hydrophilic alcohol such as methanol or ethanol, more        preferably methanol, or a hydrophilic weak organic acid,        preferably acetic acid and;    -   iii) water. The hydrophobic alcohol being the major constituent        of the mixture with multifold exess to other components. The        absorbtion composition is preferably using an        n-butanol:methanol:water or similar solvent system for        adsorption and washing the adsorbed glycans, in most preferred        system n-butanol:methanol:water in relative volumes of 10:1:2.        The preferred polar solvents for elution of the glycomes are        water or water:ethanol or similar solvent system for elution of        purified glycans from cellulose. Fractionation is possible by        using first less polar elution solvent to elute a fraction of        glycome compositions and the eluting rest by more polar solvent        such as water        3. Affinity to carbon may include purification or enrichment of        glycans due to their affinity or specific adsorption to specific        carbon materials preferably graphitized carbon, or removal of        contaminants away from the glycans. Preferred graphitized carbon        affinity methods includes porous graphitized carbon        chromatography.

Preferred purification methods according to the invention includecombinations of one or more prepurification and/or purification steps.The preferred method include preferably at least two and more preferablyat least three prepurification steps according to the invention. Thepreferred method include preferably at least one and more preferably atleast two purification steps according to the invention. It is furtherrealized that one prepurification step may be performed after apurification step or one purification step may be performed after aprepurification step. The method is preferably adjusted based on theamount of sample and impurities present in samples. Examples of thepreferred combinations include the following combinations:

For neutral underivatized glycan purification:

A. 1. precipitation and/or extraction 2. cation exchange ofcontaminants, 3. hydrophobic adsorption of contaminants, and 4.hydrophilic purification, preferably by carbon, preferably graphitizedcarbon, and/or carbohydrate affinity purification of glycans.B. 1. precipitation and/or extraction, 2. hydrophobic adsorption ofcontaminants 3. cation exchange of contaminants, 4. hydrophilicpurification by carbon, preferably graphitized carbon, and/orcarbohydrate affinity purification of glycans

The preferred method variants further includes preferred variants when

-   -   1. both carbon and carbohydrate chromatography is performed in        step 4,    -   2. only carbon chromatography is performed in step 4    -   3. only carbohydrate chromatography is performed in step 4    -   4. order steps three and four is exchanged    -   5. both precipitation and extraction are performed in        prepurification step

2) For sialylated/acidic underivatized glycan purification: The samemethods are preferred but preferably both carbon and carbohydratechromatography is performed in step 4. The carbohydrate affinitychromatography is especially preferred for acidic and/sialylatedglycans. In a preferred embodiment for additional purification one ortwo last chromatograpy methods are repeated.

D. Analysis of the Glycomes

The present invention is specifically directed to detection variouscomponent in glycomes by specific methods for recognition of suchcomponents. The methods includes binding of the glycome components byspecific binding agents according to the invention such as antibodiesand/or enzymes, these methods peferebly include labeling orimmobilization of the glycomes. For effective analysis of the glycome alarge panel of the binding agents are needed.

The invention is specifically directed to physicochemical profilingmethods for exact analysis of glycomes. The preferred methods includesmass spectrometry and NMR-spectroscopy, which give simultaneouslyinformation of numerous components of the glycomes. In a preferredembodiment the mass spectrometryand NMR-spectroscopy methods are used ina combination.

E. Quantitative and Qualitative Analysis of Glycome Data

The invention revealed methods to create reproducible and quantitativeprofiles of glycomes over large mass ranges and degrees ofpolymerization of glycans. The invention further reveals novel methodsfor quantitative analysis of the glycomics data produced by massspectrometry. The invention is specifically directed to the analysis ofnon-derivatized or reducing end derivatized glycomes according to theinvention and the glycomes containing specific structurealcharacteristics according to the invention.

The invention revealed effective means of comparision of glycomeprofiles from different cell types or cells with difference in cellstatus or cell types. The invention is especially directed to thequantitative comparision of relative amount of individual glycan signalor groups of glycan signals described by the invention.

The invention is especially directed to

i) calculating average value and variance values of signal or signals,which have obtained from several experiments/samples and whichcorrespond to an individual glycan or glycan group according to theinvention for a first cell sample and for a second cell sampleii) comparing these with values derived for the corresponding signal(s)iii) optionally calculating statistic value for testing the probabilityof similarity of difference of the values obtained for the cell types orestimating the similarity or difference based on the difference of theaverage and optionally also based on the variance values.iv) preferably repeating the comparision one or more signals or signalgroups, and further preferably performing combined statistical analysisto estimate the similarity and/or differences between the data set orestimating the difference or similarityv) preferably use of the data for estimating the differences between thefirst and second cell samples indicationg difference in cell statusand/or cell type

The invention is further directed to combining information of severalquantitative comparisions of between corresponding signals by method of

i) calculating differences between quantitative values of correspondingmost different glycan signals or glycan group signals, changing negativevalues to corresponding positive values, optionally multiplying selectedsignals by selected factors to adjust the weight of the signals in thecalculationii) adding the positive difference values to a sum valueiii) comparing the sum values as indicators of cell status or type.

It was further revealed that there is characteric signals that arepresent in certain cell types according to the invention but absent orpractically absent in other cell types. The invention is thereforedirected to the qualitative comparision of relative amount of individualglycan signal or groups of glycan signals described by the invention andobserving signals present or absent/practically absent in a cell type.The invention is further directed to selection of a cut off value usedfor selecting absent or practically absent signals from a massspectrometric data, for example the preferred cut off value may beselected in range of 0-3% of relative amount preferably the cut offvalue is less than 2%, or less than 1% or less than 0.5%. In a preferredembodiment the cut off value is adjusted or defined based on quality ofthe mass spectrum obtained, preferably based on the signal intensitiesand/or based on the number of signals observable.

The invention is further directed to automized qualitative and/orquantitative comparisions of data from corresponding signals fromdifferent samples by computer and computer programs prosessing glycomedata produced according to the invention. The invention is furtherdirected to raw data based analysis and neural network based learningsystem analysis as methods for revealing differences between the glycomedata according to the invention.

Identification and Classification of Differences in Glycan Datasets

The present invention is specifically directed to analyzing glycandatasets and glycan profiles for comparison and characterization ofdifferent cell types. In one embodiment of the invention, glycan signalsor signal groups associated with given cell type are selected from thewhole glycan datasets or profiles and indifferent glycan signals areremoved. The resulting selected signal groups have reduced backgroundand less observation points, but the glycan signals most important tothe resolving power are included in the selection. Such selected signalgroups and their patterns in different sample types serve as a signaturefor the identification of the cell type and/or glycan types orbiosynthetic groups that are typical to it. By evaluating multiplesamples from the same cell type, glycan signals that have individuali.e. cell line specific variation can be excluded from the selection.Moreover, glycan signals can be identified that do not differ betweencell types, including major glycans that can be considered ashousekeeping glycans.

To systematically analyze the data and to find the major glycan signalsassociated with given cell type according to the invention,difference-indicating variables can be calculated for the comparison ofglycan signals in the glycan datasets. Preferential variables betweentwo samples include variables for absolute and relative difference ofgiven glycan signal between the datasets from two cell types. Mostpreferential variables according to the invention are:

1. absolute difference A=(S2−S1), and2. relative difference R=A/S1,wherein S1 and S2 are relative abundances of a given glycan signal incell types 1 and 2, respectively.

It is realized that other mathematical solutions exist to express theidea of absolute and relative difference between glycan datasets, andthe above equations do not limit the scope of the present invention.According to the present invention, after A and R are calculated for theglycan profile datasets of the two cell types, the glycan signals arethereafter sorted according to the values of A and R to identify themost significant differing glycan signals. High value of A or Rindicates association with cell type 2, and vice versa. In the list ofglycan data sorted independently by R and A, the cell-type specificglycans occur at the top and the bottom of the lists. Morepreferentially, if a given signal has high values of both A and R, it ismore significant.

Preferred Representation of the Dataset when Comparing Two CellMaterials

The present invention is specifically directed to the comparativepresentation of the quantitative glycome dataset as multidimensionalgraphs comparing the paraller data for example as shown in FIGS. 41 and42 or as other three dimensional presentations or for example as twodimensional matrix showing the quantities with a quantitative code,preferably by a quantitative color code.

Released Glycomes

The invention is directed to methods to produce released, in a preferredenzymatically released glycans, also referred as glycomes, fromembryonal type cells. A preferred glycome type is N-glycan glycomereleased by a N-glycosidase enzyme. The invention is further directed toprofiling analysis of the released glycomes.

Low Amounts of Cells for Glycome Analysis from Stem Cells

The invention revealed that its possible to produce glycome from verylow amount of cells. The preferred embodiments amount of cells isbetween 1000 and 10 000 000 cells, more preferably between 10 000 and 1000 000 cells. The invention is further directed to analysis of releasedglycomes of amount of at least 0.1 pmol, more preferably of at least to1 pmol more preferably at least of 10 pmol.

(a) Total asparagine-linked glycan (N-glycan) pool was enzymaticallyisolated from about 100 000 cells. (b) The total N-glycan pool (picomolequantities) was purified with microscale solid-phase extraction anddivided into neutral and sialylated N-glycan fractions. The N-glycanfractions were analyzed by MALDI-TOF mass spectrometry either inpositive ion mode for neutral N-glycans (c) or in negative ion mode forsialylated glycans (d). Over one hundred N-glycan signals were detectedfrom each cell type revealing the surprising complexity of hESCglycosylation. The relative abundances of the observed glycan signalswere determined based on relative signal intensities (Saarinen et al.,1999, Eur. J. Biochem. 259, 829-840).

Methods for Low Sample Amounts

The present invention is specifically directed to methods for analysisof low amounts of samples.

The invention further revealed that it is possible to use the methodsaccording to the invention for analysis of low sample amounts. It isrealized that the cell materials are scarce and difficult to obtain fromnatural sources. The effective analysis methods would spare importantcell materials. Under certain circumstances such as in context of cellculture the materials may be available from large scale. The requiredsample scale depends on the relative abundancy of the characteristiccomponents of glycome in comparision to total amount of carbohydrates.It is further realized that the amount of glycans to be measured dependon the size and glycan content of the cell type to be measured andanalysis including multiple enzymatic digestions of the samples wouldlikely require more material. The present invention revealed especiallyeffective methods for free released glycans.

The picoscale samples comprise preferably at least about 1000 cells,more preferably at least about 50 000 cells, even more more preferablyat least 100 000 cells, and most preferably at least about 500 000cells. The invention is further directed to analysis of about 1 000 000cells. The preferred picoscale samples contain from at least about 1000cells to about 10 000 000 cells according to the invention. The usefulrange of amounts of cells is between 50 000 and 5 000 000, even morepreferred range of cells is between 100 000 and 3 000 000 cells. Apreferred practical range for free oligosaccharide glycoomes is betweenabout 500 000 and about 2 000 000 cells. It is realized that cellcounting may have variation of less than 20%, more preferably 10% andmost preferably 5%, depending on cell counting methods and cell sample,these variations may be used instead of term about. It is furtherunderstood that the methods according to the present invention can beupscaled to much larger amounts of material and the pico/nanoscaleanalysis is a specific application of the technology.

The invention is specifically directed to use of microcolumntechnologies according to the invention for the analysis of thepreferred picoscale and low amount samples according to the invention,

The invention is specifically directed to purification to level, whichwould allow production of high quality mass spectrum covering the broadsize range of glycans of glycome compositions according to theinvention.

Glycan Preparation and Purification for Glycome Analysis of CellMaterials According to the Invention, Especially for Mass SpectrometricMethods Use of Microfluidistic Methods Including MicrocolumnChromatography

The present invention is especially directed to use microfluidisticmethods involving low sample volumes in handling of the glycomes in lowvolume cell preparation, low volume glycan release and variouschromatographic steps. The invention is further directed to integratedcell preparation, glycan release, and purification and analysis steps toreduce loss of material and material based contaminations. It is furtherrealized that special cleaning of materials is required for optimalresults.

Low Volume Reaction in Cell Preparation and Glycan Release

The invention is directed to reactions of volume of 1-100 microliters,preferably about 2-50 microliters and even more preferably 3-20microliters, most preferably 4-10 microliter. The most preferredreaction volumes includes 5-8 microliters+/−1 microliters. The minimumvolumes are preferred to get optimally concentrated sample forpurification. The amount of material depend on number of experiment inanalysis and larger amounts may be produced preferably when multiplestructural analysis experiments are needed.

It is realized that numerous low volume chromatographic technologies maybe applied, such low volume column and for example disc basedmicrofluidistic systems.

The inventors found that the most effective methods are microcolumns.Small colomn can be produced with desired volume. Preferred volumes ofmicrocolumns are from about 2 Microliters to about 500 microliters, morepreferably for rutine sample sizes from about 5. microliter to about 100microliters depending on the matrix and size of the sample. Preferredmicrocolumn volumes for graphitised carbon, cellulose chromatography andother tip-columns are from 2 to 20 μl, more preferably from 3 to 15 μl,even more preferably from 4 to 10 μl, For the microcolumn technologiesin general the samples are from about 10 000 to about million cells. Themethods are useful for production of picomol amounts of total glycomemixtures from cells according to the invention.

In a preferred embodiment microcolumns are produced in regulardisposable usually plastic pipette tips used for example in regular“Finnpipette”-type air-piston pipettes. The pipette-tip microcolumncontain the preferred chromatographic matrix. In a preferred embodimentthe microcolumn contains two chromatographic matrixes such as an anionand cation exchange matrix or a hydrophilic and hydrophobicchromatography matrix.

The pipette tips may be chosen to be a commercial tip contain a filter.In a preferred embodiment the microcolumn is produced by narrowing athin tip from lower half so that the preferred matrix is retained in thetip. The narrowed tip is useful as the volume of filter can be omittedfrom washing steps

The invention is especially directed to plastic pipette tips containingthe cellulose matrix, and in an other embodiment to the pipette tipmicroclumns when the matrix is graphitised carbon matrix. The inventionis further directed to the preferred tip columns when the columns arenarorrowed tips, more preferably with column volumes of 1 microliter to100 microliters.

The invention is further directed to the use of the tip columnscontaining any of the preferred chromatographic matrixes according tothe invention for the purification of glycomes according to theinvention, more preferably matrixes for ion exchange, especiallypolystyrene anion exchangers and cation exchangers according to theinvention; hydrophilic chromatographic matrixes according to theinvention, especially carbohydrate matrixes and most cellulose matrixes.

NMR-analysis of Glycomes

The present invention is directed to analysis of released glycomes byspectrometric method useful for characterization of the glycomes. Theinvention is directed to NMR spectroscopic analysis of the mixtures ofreleased glycans. The inventors showed that it is possible to produce areleased glycome from human stem cells in scale large enough and ofuseful purity for NMR analysis of the glycome.

The invention is especially directed to methods of producing NMR fromspecific subglycomes, preferably N-linked glycome, O-linked glycome,glycosaminoglycan glycome and/or glycolipid glycome. The NMR-profilingaccording to the invention is further directed to the analysis of thenovel and rare structure groups revealed from cell glycomes according tothe invention. The general information about complex cell glycomematerial directed NMR-methods are limited.

Preferably the NMR-analysis is performed from an isolated subglycome.The preferred isolated subglycomes include acidic glycomes and neutralglycomes.

NMR-Glycome Analysis from Larger Amounts of Cells

It is realized that numerous methods have been described forpurification of oligosaccharide mixtures useful for NMR from variousmaterials, including usually purified individual proteins. It isrealized that present methods are useful for NMR-profiling even forlarger amounts of cells according to the invention, especially incombination with NMR-profiling according to the invention and/or whendirected to the analysis specific and preferred structure groupsaccording to the invention The preferred purification methods areeffective and form an optimised process for purification of glycomesfrom even larger amounts of cells and tissues than described fornanoscale methods below. The methods are preferred also for any largeramount of cells.

Purification Method for Low Amount Nanoscale NMR-profiling of CellSamples

Moreover, when purification methods for larger amounts of carbohydratematerials exists, but very low and complex carbohydrate materials withvery complex impurities such as cell-derived materials have been lessstudied as low amounts, especially when purity useful for NMR-analysisis needed.

Preferred Sample Amounts Allowing Effective NMR Analysis of CellGlycomes

The invention specifically revealed that NMR-samples can be producedfrom very low amounts of cells according to the invention. Preferredsample amounts of cells for a one-dimensional proton-NMR profiling arefrom about 2 million to 100 million cells, more preferably 10-50 millioncells. It is further realized that good quality NMR data can be obtainedfrom samples containing at least about 10-20 million cells.

The preferred analysis methods is directed to high resolution NMRobserving oligosaccharide/saccharide conjugate mixture from an amount ofat least 4 nmol more preferably at least 1 nmol and the cell amountyielding the preferred amount of saccharide mixture. For nanoscaleanalysis according to the invention cell material is selected so that itwould yield at least about 50 nmol of oligosaccharide mixture, morepreferably at least about 5 nmol and most preferably at least about 1nmol of oligosaccharide mixture. Preferred amounts of major componentsin glycomes to be observed effectively by the methods according to theinvention include yield at least about 10 nmol of oligosaccharidecomponent, more preferably at least about 1 nmol and most preferably atleast about 0.2 mmol of oligosaccharide component.

The preferred cell amount for analysis of a subglycome from a cell typeis preferably optimised by measuring the amounts of glycans producedfrom the cell amounts of preferred ranges.

It is realized that depending on the cell and subglycome type therequired yield of glycans and the heterogeneity of the materials varyyielding different amounts of major components.

Preferred Purification Methods

For the production of sample for nanoscale NMR, the methods describedfor preparation of cell samples and release of oligosaccharides for massspectrometric profiling according to the invention may be applied.

For the purification of sample for nanoscale NMR the methods describedfor purification mass spectrometry profiling samples according to theinvention may be applied.

The preferred purification method for nanoscale NMR-profiling accordingto the invention include following general purification process steps:

1) Precipitation/extraction;

2) Hydrophobic interaction;3) Affinity to carbon material, especially graphitized carbon.4) Gel filtration chromatography

The more preferred purification process includesprecipitation/extraction aimed for removal of major non-carbohydrateimpurities by separating the impurity and the glycome fraction(s) to bepurified to different phases. Hydrophobic interaction step aims topurify the glycome components from more hydrophobic impurities as theseare bound to hydrophobic chromatography matrix and the glycomecomponents are not retained. Chromatography on graphitized carbon mayinclude purification or enrichment of glycans due to their affinity orspecific adsorption to graphitized carbon, or removal of contaminantsfrom the glycans. The glycome components obtained by the aforementionedsteps are then subjected to gel filtration chromatography, separatingmolecules according to their hydrodynamic volume, i.e. size in solution.The gel filtration chromatography step allows detection and quantitationof glycome components by absorption at low wavelengths (205-214 nm).

The most preferred purification process includesprecipitation/extraction and hydrophobic interaction steps aimed forremoval of major non-carbohydrate impurities and more hydrophobicimpurities. Chromatography on graphitized carbon is used for removal ofcontaminants from the glycans, and to devide the glycome components tofractions of neutral glycome components and acidic glycome components.The neutral and acidic glycome component fractions are then subjected togel filtration chromatography, separating molecules according to theirsize. Preferably, the neutral glycome component fraction ischromatographed in water and the acidic glycome component fraction ischromatographed in 50-200 mM aqueous ammonium bicarbonate solution. Thegel filtration chromatography step allows detection and quantitation ofglycome components by absorption at low wavelengths (205-214 nm).Fractions showing absorbance are subjected to MALDI-TOF massspectrometry.

Preferred Methods for Producing Enriched Glycome Fractions for NMR

The fractionation can be used to enrich components of low abundance. Itis realized that enrichment would enhance the detection of rarecomponents. The fractionation methods may be used for larger amounts ofcell material. In a preferred embodiment the glycome is fractionatedbased on the molecular weight, charge or binding to carbohydrate bindingagents such as lectins and/or other binding agents according to theinvention.

These methods have been found useful for specific analysis of specificsubglycomes and enrichment more rare components. The present inventionis in a preferred embodiment directed to charge based separation ofneutral and acidic glycans. This method gives for analysis method,preferably mass spectroscopy material of reduced complexity and it isuseful for analysis as neutral molecules in positive mode massspectrometry and negative mode mass spectrometry for acidic glycans.

It is realized that preferred amounts of enriched glycomeoligosacccharide mixtures and major component comprising fractions canbe produced from larger glycome preparations.

In a preferred embodiment the invention is directed to size basedfractionation methods for effective analysis of preferred classes ofglycans in glycomes. The invention is especially directed to analysis oflower abundance components with lower and higher molecular weight thanthe glycomes according to the invention. The preferred method for sizebased fractionation is gel filtration. The invention is especiallydirected to analysis of enriched group glycans of N-linked glycomespreferably including lower molecular weight fraction includinglow-mannose glycans, and one or several preferred low mannose glycangroups according to the invention.

Preferred NMR-methods

In a preferred embodiment the NMR-analysis of the stem cell glycome isone-dimensional proton-NMR analysis showing structural reporter groupsof the major components in the glycome.

Combination of NMR- and Mass Spectrometry for Glycome Analysis

The present invention is further directed to combination of the massspectrometric and NMR analysis of stem cells. The preferred methodinclude production of any mass spectrometric profile from any glycomeaccording to the invention from a cell sample according to theinvention,

optionally characterizing the glycome by other methods like glycosidasedigestion, fragmentation mass spectrometry, specific binding agents, andproduction of NMR-profile from the same sample glycome or glycomes tocompare these profiles.The Binding Methods for Recognition of Structures from Cell SurfacesRecognition of Structures from Glycome Materials and on Cell Surfaces byBinding Methods

The present invention revealed that beside the physicochemical analysisby NMR and/or mass spectrometry several methods are useful for theanalysis of the structures. The invention is especially directed to twomethods:

-   -   i) Recognition by enzymes involving binding and alteration of        structures.    -   This method alters specific glycan structures by enzymes cabable        of altering the glycan structures. The preferred enzymes        includes        -   a) glycosidase-type enzymes capable of releasing            monosaccharide units from glycans        -   b) glycosyltransferring enzymes, including            transglycosylating enzymes and glycosyltransferases        -   c) glycan modifying enzymes including sulfate and or fosfate            modifying enzymes    -   ii) Recognition by molecules binding glycans referred as the        binders    -   These molecules bind glycans and include property allowing        observation of the binding such as a label linked to the binder.        The preferred binders include        -   a) Proteins such as antibodies, lectins and enzymes        -   b) Peptides such as binding domains and sites of proteins,            and synthetic library derived analogs such as phage display            peptides    -   c) Other polymers or organic scaffold molecules mimicking the        peptide materials

The peptides and proteins are preferably recombinant proteins orcorresponding carbohydrate recognition domains derived therereof, whenthe proteins are selected from the group monoclonal antibody,glycosidase, glycosyl transferring enzyme, plant lectin, animal lectinor a peptide mimetic thereof and wherein the binder includes adetectable label structure.

Preferred Binder Molecules

The present invention revealed various types of binder molecules usefulfor characterization of cells according to the invention and morespecifically the preferred cell groups and cell types according to theinvention. The preferred binder molecules are classified based on thebinding specificity with regard to specific structures or structuralfeatures on carbohydrates of cell surface. The preferred bindersrecognize specifically more than single monosaccharide residue.

It is realized that most of the current binder molecules such as all ormost of the plant lectins are not optimal in their specificity andusually recognize roughly one or several monosaccharides with variouslinkages. Furthermore the specificities of the lectins are usually notwell characterized with several glycans of human types.

The preferred high specificity binders recognize

-   -   A) at least one monosaccharide residue and a specific bond        structure between those to another monosaccharides next        monosaccharide residue referred as MS1B1-binder,    -   B) more preferably recognizing at least part of the second        monosaccharide residue referred as MS2B1-binder,    -   C) even more preferably recognizing second bond structure and or        at least part of third mono saccharide residue, referred as        MS3B2-binder, preferably the MS3B2 recognizes a specific        complete trisaccharide structure.    -   D) most preferably the binding structure recognizes at least        partially a tetrasaccharide with three bond structures, referred        as MS4B3-binder, preferably the binder recognizes complete        tetrasaccharide sequences.

The preferred binders includes natural human and or animal, or otherproteins developed for specific recognition of glycans. The preferredhigh specificity binder proteins are specific antibodies preferablymonoclonal antibodies; lectins, preferably mammalian or animal lectins;or specific glycosyltransferring enzymes more preferably glycosidasetype enzymes, glycosyltransferases or transglycosylating enzymes.

Target Structures for Specific Binders and Examples of the BindingMolecules

Combination of Terminal Structures in Combination with Specific GlycanCore Structures

It is realized that part of the structural elements are specificallyassociated with specific glycan core structure. The recognition ofterminal structures linked to specific core structures are especiallypreferred, such high specificity reagents have capacity of recognitionalmost complete individual glycans to the level of physicochemicalcharacterization according to the invention. For example many specificmannose structures according to the invention are in general quitecharacteristic for N-glycan glycomes according to the invention. Thepresent invention is especially directed to recognition terminalepitopes.

Common Terminal Structures on Several Glycan Core Structures

The present invention revealed that there are certain common structuralfeatures on several glycan types and that it is possible to recognizecertain common epitopes on different glycan structures by specificreagents when specificity of the reagent is limited to the terminalwithout specificity for the core structure. The invention especiallyrevealed characteristic terminal features for specific cell typesaccording to the invention. The invention realized that the commonepitopes increase the effect of the recognition. The common terminalstructures are especially useful for recognition in the context withpossible other cell types or material, which do not contain the commonterminal structure in substancial amount.

Specific Preferred Structural Groups

The present invention is directed to recognition of oligosaccharidesequences comprising specific terminal monosaccharide types, optionallyfurther including a specific core structure. The preferredoligosaccharide sequences classified based on the terminalmonosaccharide structures.

1. Structures with Terminal Mannose monosaccharide

Preferred mannose-type target structures have been specificallyclassified by the invention. These include various types of high andlow-mannose structures and hybrid type structures according to theinvention.

Low or Uncharacterised Specificity Binders

preferred for recognition of terminal mannose structures includesmannose-monosaccharide binding plant lectins.

Preferred High Specific High Specificity Binders includei) Specific mannose residue releasing enzymes such as linkage specificmannosidases, more preferably an α-mannosidase or β-mannosidase.

Preferred α-mannosidases includes linkage specific α-mannosidases suchas α-Mannosidases cleaving preferably non-reducing end terminal

α2-linked mannose residues specifically or more effectively than otherlinkages, more preferably cleaving specifically Manα2-structures; orα6-linked mannose residues specifically or more effectively than otherlinkages, more preferably cleaving specifically Manα6-structures;Preferred β-mannosidases includes β-mannosidases capable of cleavingβ4-linked mannose from non-reducing end terminal of N-glycan coreManβ4GlcNAc-structure without cleaving other β-linked monosaccharides inthe glycomes.ii) Specific binding proteins recognizing preferred mannose structuresaccording to the invention. The preferred reagents include antibodiesand binding domains of antibodies (Fab-fragments and like), and otherengineered carbohydrate binding proteins. The invention is directed toantibodies recognizing MS2B1 and more preferably MS3B2-structures2. Structures with Terminal Gal-monosaccharide

Preferred galactose-type target structures have been specificallyclassified by the invention. These include various types ofN-acetyllactosamine structures according to the invention.

Low or uncharacterised Specificity Binders for Terminal Gal

Prereferred for recognition of terminal galactose structures includesplant lectins such as ricin lectin (ricinus communis agglutinin RCA),and peanut lectin(/agglutinin PNA).

Preferred High Specific High Specificity Binders Include

i) Specific galactose residue releasing enzymes such as linkage specificgalactosidases, more preferably α-galactosidase or β-galactosidase.

Preferred α-galactosidases include linkage galactosidases capable ofcleaving Galα3Gal-structures revealed from specific cell preparations

Preferred β-galactosidases includes β-galactosidases capable of cleaving

β4-linked galactose from non-reducing end terminal Galβ4GlcNAc-structurewithout cleaving other β-linked monosaccharides in the glycomes andβ3-linked galactose from non-reducing end terminal Galβ3GlcNAc-structurewithout cleaving other C-linked monosaccharides in the glycomesii) Specific binding proteins recognizing preferred galactose structuresaccording to the invention. The preferred reagents include antibodiesand binding domains of antibodies (Fab-fragments and like), and otherengineered carbohydrate binding proteins and animal lectins such asgalectins.3. Structures with Terminal GalNAc-monosaccharide

Preferred GalNAc-type target structures have been specifically revealedby the invention. These include especially LacdiNAc, GalNAcβGlcNAc-typestructures according to the invention.

Low or Uncharacterised Specificity Binders for Terminal GalNAc

Several plant lectins has been reported for recognition of terminalGalNAc. It is realized that some GalNAc-recognizing lectins may beselected for low specificity reconition of the preferredLacdiNAc-structures.

Preferred High Specific High Specificity Binders Include

i) The invention revealed that β-linked GalNAc can be recognized byspecific β-N-acetylhexosaminidase enzyme in combination withβ-N-acetylhexosaminidase enzyme. This combination indicates the terminalmonosaccharide and at least part of the linkage structure.

Preferred β-N-acetylehexosaminidase, includes enzyme capable of cleavingβ-linked GalNAc from non-reducing end terminal GalNAcβ4/3-structureswithout cleaving α-linked HexNAc in the glycomes; preferredN-acetylglucosaminidases include enzyme capable of cleaving β-linkedGlcNAc but not GalNAc.

ii) Specific binding proteins recognizing preferred GalNAcβ4, morepreferably GalNAcβ4GlcNAc, structures according to the invention. Thepreferred reagents include antibodies and binding domains of antibodies(Fab-fragments and like), and other engineered carbohydrate bindingproteins, and a special plant lectin WFA (Wisteria floribundaagglutinin).4. Structures with Terminal GlcNAc-monosaccharide

Preferred GlcNAc-type target structures have been specifically revealedby the invention. These include especially GlcNAcβ-type structuresaccording to the invention.

Low or Uncharacterised Specificity Binders for Terminal GlcNAc

Several plant lectins has been reported for recognition of terminalGlcNAc. It is realized that some GlcNAc-recognizing lectins may beselected for low specificity reconition of the preferredGlcNAc-structures.

Preferred High Specific High Specificity Binders Include

-   -   i) The invention revealed that β-linked GlcNAc can be recognized        by specific β-N-acetylglucosaminidase enzyme.

Preferred β-N-acetylglucosaminidase includes enzyme capable of cleavingβ-linked GlcNAc from non-reducing end terminal GlcNAcβ2/3/6-structureswithout cleaving β-linked GalNAc or α-linked HexNAc in the glycomes;

ii) Specific binding proteins recognizing preferred GlcNAcβ2/3/6, morepreferably GlcNAcβ2Manα structures according to the invention. Thepreferred reagents include antibodies and binding domains of antibodies(Fab-fragments and like), and other engineered carbohydrate bindingproteins.5. Structures with Terminal Fucose-monosaccharide

Preferred fucose-type target structures have been specificallyclassified by the invention. These include various types ofN-acetyllactosamine structures according to the invention.

Low or Uncharacterised Specificity Binders for Terminal Fuc

Prereferred for recognition of terminal fucose structures includesfucose monosaccharide binding plant lectins. Lectins of Ulex europeausand Lotus tetragonolobus has been reported to recognize for exampleterminal Fucoses with some specificity binding for α2-linked structures,and branching α3-fucose, respectively.

Preferred High Specific High Specificity Binders Include

i) Specific fucose residue releasing enzymes such as linkagefucosidases, more preferably α-fucosidase.

Preferred α-fucosidases include linkage fucosidases capable of cleavingFucα2Gal-, and Galβ4/3(Fucα3/4)GlcNAc-structures revealed from specificcell preparations.

ii) Specific binding proteins recognizing preferred fucose structuresaccording to the invention. The preferred reagents include antibodiesand binding domains of antibodies (Fab-fragments and like), and otherengineered carbohydrate binding proteins and animal lectins such asselectins recognizing especially Lewis type structures such as Lewis x,Galβ4(Fucα3)GlcNAc, and sialyl-Lewis x, SAα3Galβ4(Fucα3)GlcNAc.

The preferred antibodies includes antibodies recognizing specificallyLewis type structures such as Lewis x, and sialyl-Lewis x. Morepreferably the Lewis x-antibody is not classic SSEA-1 antibody, but theantibody recognizes specific protein linked Lewis x structures such asGalβ4(Fucα3)GlcNAcβ2Manα-linked to N-glycan core.

6. Structures with Terminal Sialic Acid-monosaccharide

Preferred sialic acid-type target structures have been specificallyclassified by the invention.

Low or Uncharacterised Specificity Binders for Terminal Fuc

Preferred for recognition of terminal sialic acid structures includessialic acid monosaccharide binding plant lectins.

Preferred High Specific High Specificity Binders Include

i) Specific sialic acid residue releasing enzymes such as linkagesialidases, more preferably α-sialidases.

Preferred α-sialidases include linkage sialidases capable of cleavingSAα3Gal- and SAα6Gal-structures revealed from specific cell preparationsby the invention.

Preferred lectins, with linkage specificity include the lectins, thatare specific for SAα3Gal-structures, preferably being Maackia amurensislectin and/or lectins specific for SAα6Gal-structures, preferably beingSambucus nigra agglutinin.

ii) Specific binding proteins recognizing preferred sialic acidoligosaccharide sequence structures according to the invention. Thepreferred reagents include antibodies and binding domains of antibodies(Fab-fragments and like), and other engineered carbohydrate bindingproteins and animal lectins such as selectins recognizing especiallyLewis type structures such as sialyl-Lewis x, SAα3Galβ4(Fucα3)GlcNAc orsialic acid recognizing Siglec-proteins.

The preferred antibodies includes antibodies recognizing specificallysialyl-N-acetyllactosamines, and sialyl-Lewis x.

Preferred antibodies for NeuGc-structures includes antibodies recognizesa structure NeuGcα3Galβ4Glc(NAc)_(0 or 1) and/orGalNAcβ4[NeuGcα3]Galβ4Glc(NAc)_(0 or 1), wherein [ ] indicates branch inthe structure and ( )_(0 or 1) a structure being either present orabsent. In a preferred embodiment the invention is directed recognitionof the N-glycolyl-Neuraminic acid structures by antibody, preferably bya monoclonal antibody or human/humanized monoclonal antibody. Apreferred antibody contains the variable domains of P3-antibody.

Binder-label Conjugates

The present invention is specifically directed to the binding of thestructures according to the present invention, when the binder isconjugated with “a label structure”. The label structure means amolecule observable in a assay such as for example a fluorescentmolecule, a radioactive molecule, a detectable enzyme such as horseradish peroxidase or biotin/streptavidin/avidin. When the labelledbinding molecule is contacted with the cells according to the invention,the cells can be monitored, observed and/or sorted based on the presenceof the label on the cell surface. Monitoring and observation may occurby regular methods for observing labels such as fluorescence measuringdevices, microscopes, scintillation counters and other devices formeasuring radioactivity.

Use of Binder and Labelled Binder-conjugates for Cell Sorting

The invention is specifically directed to use of the binders and theirlabelled cojugates for sorting or selecting human stem cells frombiological materials or samples including cell materials comprisingother cell types. The preferred cell types includes cord blood,peripheral blood and embryonal stem cells and associated cells. Thelabels can be used for sorting cell types according to invention fromother similar cells. In another embodiment the cells are sorted fromdifferent cell types such as blood cells or in context of cultured cellspreferably feeder cells, for example in context of embryonal stem cellscorresponding feeder cells such as human or mouse feeder cells. Apreferred cell sorting method is FACS sorting. Another sorting methodsutilized immobilized binder structures and removal of unbound cells forseparation of bound and unbound cells.

Use of Immobilized Binder Structures

In a preferred embodiment the binder structure is conjugated to a solidphase. The cells are contacted with the solid phase, and part of thematerial is bound to surface. This method may be used to separation ofcells and analysis of cell surface structures, or study cell biologicalchanges of cells due to immobilization. In the analytics involvingmethod the cells are preferably tagged with or labelled with a reagentfor the detection of the cells bound to the solid phase through a binderstructure on the solid phase. The methods preferably further include oneor more steps of washing to remove unbound cells.

Preferred solid phases include cell suitable plastic materials used incontacting cells such as cell cultivation bottles, petri dishes andmicrotiter wells; fermentor surface materials

Specific Recognition between Preferred Stem Cells and ContaminatingCells

The invention is further directed to methods of recognizing stem cellsfrom differentiated cells such as feeder cells, preferably animal feedercells and more preferably mouse feeder cells. It is further realized,that the present reagents can be used for purification of stem cells byany fractionation method using the specific binding reagents.

Preferred fractionation methods includes fluorecense activated cellsorting (FACS), affinity chromatography methods, and bead methods suchas magnetic bead methods.

Preferred reagents for recognition between preferred cells, preferablyembryonal type cells, and contaminating cells, such as feeder cells,most preferably mouse feeder cells, include reagents according to theTable 49, more preferably proteins with similar specificity with lectinsPSA, MAA, and PNA.

The invention is further directed to positive selection methodsincluding specific binding to the stem cell population but not tocontaminating cell population. The invention is further directed tonegative selection methods including specific binding to thecontaminating cell population but not to the stem cell population. Inyet another embodiment of recognition of stem cells the stem cellpopulation is recognized together with a homogenous cell population suchas a feeder cell population, preferably when separation of othermaterials is needed. It is realized that a reagent for positiveselection can be selected so that it binds stem cells as in the presentinvention and not to the contaminating cell population and a reagent fornegative selection by selecting opposite specificity. In case of onepopulation of cells according to the invention is to be selected from anovel cell population not studied in the present invention, the bindingmolecules according to the invention maybe used when verified to havesuitable specificity with regard to the novel cell population (bindingor not binding). The invention is specifically directed to analysis ofsuch binding specificity for development of a new binding or selectionmethod according to the invention.

The preferred specificities according to the invention includerecognition of:

-   -   i) mannose type structures, especially alpha-Man structures like        lectin PSA, preferably on the surface of contaminating cells    -   ii) α3-sialylated structures similarity as by MAA-lectin,        preferably for recognition of embryonal type stem cells    -   iii) Gal/GalNAc binding specificity, preferably Gal1-3/GalNAc1-3        binding specificity, more preferably Galβ1-3/GalNAcβ1-3 binding        specificity similar to PNA, preferably for recognition of        embryonal type stem cells

Manipulation of Cells by Binders

The invention is specifically directed to manipulation of cells by thespecific binding proteins. It is realized that the glycans describedhave important roles in the interactions between cells and thus bindersor binding molecules can be used for specific biological manipulation ofcells. The manipulation may be performed by free or immobilized binders.In a preferred embodiment cells are used for manipulation of cell undercell culture conditions to affect the growth rate of the cells.

Preferred Cell Population to be Produced by Glycomodification Accordingto the Present Invention

The present invention is directed to specific cell populationscomprising in vitro enzymatically altered glycosylations according tothe present invention. It is realized that special structures revealedon cell surfaces have specific targeting, and immune recognitionproperties with regard to cells carrying the structures. It is realizedthat sialylated and fucosylated terminal structures such as sialyl-lewisx structures target cells to selectins involved in bone marrow homing ofcells and invention is directed to methods to produce such structures oncells surfaces. It is further realized that mannose and galactoseterminal structures revealed by the invention target cells to liverand/or to immune recognition, which in most cases are harmful foreffective cell therapy, unless liver is not targeted by the cells. NeuGcis target for immune recognition and has harmful effects for survival ofcells expressing the glycans.

The invention revealed glycosidase methods for removal of the structuresfrom cell surface while keeping the cells intact. The invention isespecially directed to sialyltransferase methods for modification ofterminal galactoses. The invention further revealed novel method toremove mannose residues from intact cells by alpha-manosidase.

The invention is further directed to metabolic regulation ofglycosylation to alter the glycosylation for reduction of potentiallyharmful structures.

The present invention is directed to specific cell populationscomprising in vitro enzymatically altered sialylation according to thepresent invention. The preferred cell population includes cells withdecreased amount of sialic acids on the cell surfaces, preferablydecreased from the preferred structures according to the presentinvention. The altered cell population contains in a preferredembodiment decreased amounts of α3-linked sialic acids. The presentinvention is preferably directed to the cell populations when the cellpopulations are produced by the processes according to the presentinvention.

Cell Populations with Altered Sialylated Structures

The invention is further directed to novel cell populations producedfrom the preferred cell populations according to the invention when thecell population comprises altered sialylation as described by theinvention. The invention is specifically directed to cell populationscomprising decreased sialylation as described by the invention. Theinvention is specifically directed to cell populations comprisingincreased sialylation of specific glycan structures as described by theinvention. Furthermore invention is specifically directed to cellpopulations of specifically altered α3- and or α6-sialylation asdescribed by the invention These cells are useful for studies ofbiological functions of the cell populations and role of sialylated,linkage specifically sialylated and non-sialylated structures in thebiological activity of the cells.

Preferred Cell Populations with Decreased Sialylation

The preferred cell population includes cells with decreased amount ofsialic acids on the cell surfaces, preferably decreased from thepreferred structures according to the present invention. The alteredcell population contains in a preferred embodiment decreased amounts ofα3-linked sialic or α6-linked sialic acid. In a preferred embodiment thecell populations comprise practically only α3-sialic acid, and inanother embodiment only α6-linked sialic acids, preferably on thepreferred structures according to the invention, most preferably on thepreferred N-glycan structures according to the invention. The presentinvention is preferably directed to the cell populations when the cellpopulations are produced by the processes according to the presentinvention. The cell populations with altered sialylation are preferablymesenchymal stem cell, embryonal-type cells or cord blood cellpopulations according to the invention.

Preferred Cell Populations with Increased Sialylation

The preferred cell population includes cells with increased amount ofsialic acids on the cell surfaces, preferably decreased from thepreferred structures according to the present invention. The alteredcell population contains in preferred embodiments increased amounts ofα3-linked sialic or α6-linked sialic acid in a preferred embodiment thecell populations comprise practically only α3-sialic acid, and inanother embodiment only α6-linked sialic acids, preferably on thepreferred structures according to the invention, most preferably on thepreferred N-glycan structures according to the invention. The presentinvention is preferably directed to the cell populations when the cellpopulations are produced by the processes according to the presentinvention. The cell populations with altered sialylation are preferablymesenchymal stem cells or embryonal-type cells or cord blood cellpopulations according to the invention.

Preferred Cell Populations with Altered Sialylation

The preferred cell population includes cells with altered linkagestructures of sialic acids on the cell surfaces, preferably decreasedfrom the preferred structures according to the present invention. Thealtered cell population contains in a preferred embodiments alteredamount of α3-linked sialic and/or α6-linked sialic acid. The inventionis specifically directed to cell populations having a sialylation levelsimilar to the original cells but the linkages of structures are alteredto α3-linkages and in another embodiment the linkages of structures arealtered to α6-structures. In a preferred embodiment the cell populationscomprise practically only α3-sialic acid, and in another embodiment onlyα6-linked sialic acids, preferably on the preferred structures accordingto the invention, most preferably on the preferred N-glycan structuresaccording to the invention. The present invention is preferably directedto the cell populations when the cell populations are produced by theprocesses according to the present invention. The cell populations withaltered sialylation are preferably mesenchymal stem cells orembryonal-type cells or cord blood cell populations according to theinvention.

Cell Populations Comprising Preferred Cell Populations with PreferredSialic Acid Types

The preferred cell population includes cells with altered types ofsialic acids on the cell surfaces, preferably on the preferredstructures according to the present invention. The altered cellpopulation contains in a preferred embodiment altered amounts of NeuAcand/or NeuGc sialic acid. The invention is specifically directed to cellpopulations having sialylation levels similar to original cells but thesialic acid structures altered to NeuAc and in another embodiment thesialic acid type structures altered to NeuGc. In a preferred embodimentthe cell populations comprise practically only NeuAc, and in anotherembodiment only NeuGc sialic acids, preferably on the preferredstructures according to the invention, most preferably on the preferredN-glycan structures according to the invention. The present invention ispreferably directed to the cell populations when the cell populationsare produced by the processes according to the present invention. Thecell populations with altered sialylation are preferably mesenchymalstem cells or embryonal-type cells or cord blood cell populationsaccording to the invention.

Methods to Alter (Remove or Reduce or Change) Glycosylation of CellsAnalysis and Degradative Removal of the Harmful Glycan Structure

The present invention is further directed to degradative removal ofspecific harmful glycan structures from cell, preferably from desiredcell populations according to the invention.

The removal of the glycans or parts thereof occur preferably by enzymessuch as glycosidase enzymes.

In some cases the removal of carbohydrate structure may reveal anotherharmful structure. In another preferred embodiment the present inventionis directed to replacement of the removed structure by less harmful orbetter tolerated structure more optimal for the desired use.

Desialylation Methods Preferred Special Target Cell Type

Effective and specific desialylation methods for the specific cellpopulations were developed. The invention is specifically directed todesialylation methods for modification of human cord blood cells. Thecord blood cells are clearly different of other cell types and nodesialylation methods have previously been developed for these cells.Due to cell specific differences any quantitative desialylation methodscannot be generalized from one cell population to another. Thus, anyresults and data demonstrated by other investigators using other celltypes are not applicable to cord blood. The present invention is furtherdirected to desialylation modifications of any human stem cell or cordblood cell subpopulation.

The present invention is specifically directed to methods fordesialylation of the preferred structures according to the presentinvention from the surfaces of preferred cells. The present invention isfurther directed to preferred methods for the quantitative verificationof the desialylation by the preferred analysis methods according to thepresent invention. The present invention is further directed to linkagespecific desialylation and analysis of the linkage specific sialylationon the preferred carbohydrate structures using analytical methodsaccording to the present invention

The invention is preferably directed to linkage specificα3-desialylation of the preferred structures according to the inventionwithout interfering with the other sialylated structures according tothe present invention. The invention is further directed to simultaneousdesialylation α3- and α6-sialylated structures according to the presentinvention.

Furthermore the present invention is directed to desialylation when bothNeuAc and NeuGc are quantitatively removed from cell surface, preferablyfrom the preferred structures according to the present invention. Thepresent invention is specifically directed to the removal of NeuGc frompreferred cell populations, most preferably cord blood and stem cellpopulations and from the preferred structures according to the presentinvention. The invention is further directed to preferred methodsaccording to the present invention for verification of removal of NeuGc,preferably quantitative verification and more preferably verificationperformed by mass spectrometry.

Modification of Cell Surfaces of the Preferred Cells byGlycosyltrasferases

The inventors revealed that it is possible to produce controlled cellsurface glycosylation modifications on the preferred cells according tothe invention. The present invention is specifically directed toglycosyltransferase catalysed modifications of N-linked glycans on thesurfaces of cells, preferably blood cells, more preferably leukocytes orstem cells or more preferably the preferred cells according to thepresent invention.

The present invention is directed to cell modifications bysialyltransferases and fucosyltransferases. Two most preferred transferreactions according to the invention are α3-modification reactions suchas α3-sialylation and α3-fucosylations. When combined these reactionscan be used to produce important cell adhesion structures which aresialylated and fucosylated N-acetyllactosamines such as sialyl-Lewis x(sLex).

Sialylation

Possible α6-sialylation has been implied in bone marrow cells and inperipheral blood CD34+ cells released from bone marrow to circulation bygrowth factor administration, cord blood cells or other stem cell typeshave not been investigated. Furthermore, the previous study utilized anartificial sialic acid modification method, which may affect thespecificity of the sialyltransferase enzyme and, in addition, the actualresult of the enzyme reaction is not known as the reaction products werenot analysed by the investigators. The reactions are likely to have beenvery much limited by the specificity of the α6-sialyltransferase usedand cannot be considered prior art in respect to the present invention.

The inventors of the present invention further revealed effectivemodification of the preferred cells according to the present inventionsby sialylation, in a preferred embodiment by α3-sialylation.

The prior art data cited above does not indicate the specificmodifications according to the present invention to cells from earlyhuman blood, preferably cord blood, to cultured mesenchymal stem cells,or to cultured embryonal type cells. The present invention isspecifically directed to sialyltransferase reactions towards these celltypes. The invention is directed to sialyltransferase catalyzed transferof a natural sialic acid, preferably NeuAc, NeuGc or Neu-O-Ac, fromCMP-sialic acid to target cells.

Sialyltransferase catalyzed reaction according to Formula:

CMP-SA+target cell→SA-target cell+CMP,

Wherein SA is a sialic acid, preferably a natural sialic acid,preferably NeuAc, NeuGc or Neu-O-Ac andthe reaction is catalysed by a sialyltransferase enzyme preferably by anα3-sialyltransferaseandthe target cell is a cultured stern cell or early human blood cell (cordblood cell).

Preferably the sialic acid is transferred to at least one N-glycanstructure on the cell surface, preferably to form a preferred sialylatedstructure according to the invention

Fucosyltransferase Reactions

In the prior art fucosyltransferase reactions towards unspecified cellsurface structures has been studied

The prior art indicates that human cord blood cell populations may beα3-fucosylated by human fucosyltransferase VI and such modified cellpopulations may be directed to bone marrow due to interactions withselecting.

Directing Cells and Selectin Ligands

The present invention describes reactions effectively modifying cordblood cells by fucosyltransferases, especially in order to producesialylated and fucosylated N-acetyllactosamines on cell surfaces,preferably sLex and related structures. The present invention is furtherdirected to the use of the increased sialylated and/or fucosylatedstructures on the cell surfaces for targeting the cells, in a preferredembodiment for selectin directed targeting of the cells.

The invention is further directed to α3- and/or α4-fucosylation ofcultured stem cells, preferably embryonal stem cells and mesenchymalstem cells derived either from cord blood or bone marrow.

Fucosylation of Human Peripheral Blood Mononuclear Cell Populations

In a specific embodiment the present invention is directed toα3-fucosylation of the total mononuclear cell populations from humanperipheral blood. Preferably the modification is directed to at least toone protein linked glycan, more preferably to a N-linked glycan. Theprior art reactions reported about cord blood did not describe reactionsin such cell populations and the effect of possible reaction cannot beknown. The invention is further directed to combined increasedα3-sialylation and fucosylation, preferably α3-sialylation of humanperipheral blood leukocytes. It is realized that the structures on theperipheral blood leukocytes can be used for targeting the peripheralblood leukocytes, preferably to selecting expressing sites such asselectin expressing malignant tissues.

Methods for Combined Increased α3-sialylation and α3-fucosylation

The invention is specifically directed to selection of a cell populationfrom the preferred cell population according to the present invention,when the cell population demonstrate increased amount of α3-sialylationwhen compared with the baseline cell populations.

The inventors revealed that human cord blood in general is highlyα6-sialylated and thus not a good target for α3/4-fucosylationreactions, especially for reactions directed to production of selectinligand structures.

Use of Selected Cultured α3-sialic Acid Expressing Cell Populations

The inventors revealed that specific subpopulations of native cord bloodcells express increased amounts of α3-linked sialic acid. Preferredselected cell populations from cord blood for α3/4-fucosylation includeCD133+ cells.

Furthermore it was found that cultured cells according to the inventionhave a high tendency to express α3-sialic acid instead to α6-linkedsialic acids. The present invention is preferably directed to culturedmesenchymal stem cell lines, more preferably mesenchymal stem cells frombone marrow or from cord blood expressing increased amounts of α3-linkedsialic acid

Fucosylation of α3-sialylated Cells

The present invention is preferably directed to fucosylation afterα3-sialylation of cells, preferably the preferred cells according to theinvention. The invention describes for the first time combined reactionby two glycosyltransferases for the production of specific terminalepitopes comprising two different monosaccharide types on cell surfaces.

Fucosylation of Desialylated and α3-sialylated Cells

The present invention is preferably directed to fucosylation afterdesialylation and β3-sialylation of cells, preferably the preferredcells according to the invention. The invention describes for the firsttime combined reaction by two glycosyltransferases and a glycosidase forthe production of specific terminal epitopes comprised of two differentmonosaccharide types on cell surfaces.

Sialylation Methods Preferred Special Target Cell Type Early Human Blood

Effective specific sialylation methods for the specific cell populationswere developed. The invention is specifically directed to sialylationmethods for modification of human cord blood cells and subpopulationsthereof and multipotent stem cell lines. The cord blood cells areclearly different from other cell types and no sialylation methods havebeen developed for the cell population. Due to cell specific differencesany quantitative sialylation methods cannot be generalized from one cellpopulation to another. The present invention is further directed tosialylation modifications of any human cord blood cell subpopulation.

Embryonal-type Cells and Mesenchymal Stem Cells

The methods of present invention are further directed to the methodsaccording to the invention for altering human embryonal-type andmesenchymal stem cells. In a preferred embodiment the modificationtechnologies is directed to cultured cells according to the invention.

Production of Preferred Sialylated Structures

Present invention is specifically directed to methods for sialylation toproduce preferred structures according to the present invention from thesurfaces of preferred cells. The present invention is specificallydirected to production preferred NeuGc- and NeuAc-structures. Theinvention is directed to production of potentially in vivo harmfulstructures on cells surfaces, e.g. for control materials with regard tocell labelling. The invention is further directed to production ofspecific preferred terminal structure types, preferably α3- andα6-sialylated structures, and specifically NeuAc- and NeuGc-structuresfor studies of biological activities of the cells.

The present invention is further directed to preferred methods for thequantitative verification of the sialylation by the preferred analysismethods according to the present invention. The present invention isfurther directed to linkage specific sialylation and analysis of thelinkage specific sialylation on the preferred carbohydrate structuresusing analytical methods according to the present invention.

The invention is preferably directed to linkage specific α3-sialylationof the preferred structures according to the invention withoutinterfering with the other sialylated structures according to thepresent invention. The invention is preferably directed to linkagespecific α6-sialylation of the preferred structures according to theinvention without interfering with the other sialylated structuresaccording to the present invention.

The invention is further directed to simultaneous sialylation α3- andα6-sialylated structures according to the present invention. The presentinvention is further directed for the production of preferred relationof α3- and α6-sialylated structures, preferably in single reaction withtwo sialyl-transferases.

Furthermore the present invention is directed to sialylation when eitherNeuAc or NeuGc are quantitatively synthesized to the cell surface,preferably on the preferred structures according to the presentinvention. Furthemmore the invention is directed to sialylation whenboth NeuAc and NeuGc are, preferably quantitatively, transferred toacceptor sites on the cell surface.

The present invention is specifically directed to the removal of NeuGcfrom preferred cell populations, most preferably cord blood cellpopulations and from the preferred structures according to the presentinvention, and resialylation with NeuAc.

The invention is further directed to preferred methods according to thepresent invention for verification of removal of NeuGc, andresialylation with NeuAc, preferably quantitative verification and morepreferably verification performed by mass spectrometry with regard tothe preferred structures.

Controlled Cell Modification

The present invention is further directed to cell modification accordingto the invention, preferably desialylation or sialylation of the cellsaccording to the invention, when the sialidase reagent is a controlledreagent with regard of presence of carbohydrate material.

Purification of Cells with Regard to Modification Enzyme

The preferred processes according to the invention comprise of the stepof removal of the enzymes from the cell preparations, preferably thesialyl modification enzymes according to the invention. Most preferablythe enzymes are removed from a cell population aimed for therapeuticuse. The enzyme proteins are usually antigenic, especially when theseare from non-mammalian origin. If the material is not of human originits glycosylation likely increases the antigenicity of the material.This is particularity the case when the glycosylation has majordifferences with human glycosylation, preferred examples of largelydifferent glycosylations includes: procaryotic glycosylation, plant typeglycosylation, yeast or fungal glycosylation, mammalian/animalglycosylation with Galα3Galβ4GlcNAc-structures, animal glycosylationswith NeuGc structures. The glycosylation of a recombinant enzyme dependson the glycosylation in the production cell line, these producepartially non-physiological glycan structures. The enzymes arepreferably removed from any cell populations aimed for culture orstorage or therapeutic use. The presence of enzymes which have affinitywith regard to cell surface may otherwise alter the cells as detectableby carbohydrate binding reagents or mass spectrometric or other analysisaccording to the invention and cause adverse immunological responses.

Under separate embodiment the cell population is cultured or stored inthe presence of the modification enzyme to maintain the change in thecell surface structure, when the cell surface structures are recoveringfrom storage especially at temperatures closer physiological or culturetemperatures of the cells. Preferably the cells are then purified fromtrace amounts of the modification enzyme before use.

The invention is furthermore directed to methods of removal of themodification reagents from cell preparations, preferably themodification reagents are desialylation or resialylation reagents. It isrealized that soluble enzymes can be washed from the modified cellpopulations. Preferably the cell material to be washed is immobilized ona matrix or centrifuged to remove the enzyme, more preferablyimmobilized on a magnetic bead matrix.

However, extraneous washing causes at least partial destruction of cellsand their decreased viability. Furthermore, the enzymes have affinitywith regard to the cell surface. Therefore the invention is specificallydirected to methods for affinity removal of the enzymes. The preferredmethod includes a step of contacting the modified cells with an affinitymatrix binding the enzyme after modification of the cells.

Under specific embodiment the invention is directed to methods oftagging the enzyme to be removed from the cell population. The taggingstep is performed before contacting the enzyme with the cells. Thetagging group is designed to bind preferably covalently to the enzymesurface, without reduction or without major reduction of the enzymeactivity. The invention is further directed to the removal of the taggedenzyme by binding the tag to a matrix, which can be separated from thecells. Preferably the matrix comprises at least one matrix materialselected from the group: polymers, beads, magnetic beads, or solid phasesurface

Enzymes Acceptable for Humans for Modification of Reagents or Cells

Under specific embodiment the invention is directed to the use formodification of the cells according to the invention, or in a separateembodiment reagents for processes according to the invention, of a humanacceptable enzyme, preferably a glycosidase according to the inventionor in preferred embodiment sialidase or sialyltmmsferase, which isacceptable at least in certain amounts to human beings without causingharmful allergic or immune reactions. It is realized that the humanacceptable enzymes may not be needed to be removed from reactionmixtures or less washing steps are needed for desirable level of theremoval. The human acceptable enzyme is in preferred embodiment a humanglycosyltransferase or glycosidase. The present invention is separatelydirected to human acceptable enzyme which is a sialyltransferase. Thepresent invention is separately directed to human acceptable enzymewhich is a sialidase, the invention is more preferably directed to humansialidase which can remove specific type of sialic acid from cells.

In a preferred embodiment the human acceptable enzyme is purified fromhuman material, preferably from human serum, urine or milk. In anotherpreferred embodiment the enzyme is recombinant enzyme corresponding tonatural human enzyme. More preferably the enzyme corresponds to humannatural enzyme corresponds to natural cell surface or a secreted from ofthe enzyme, more preferably serum or urine or human milk form of theenzyme. Even more preferably the present invention is directed to humanacceptable enzyme which corresponds to a secreted form of a humansialyltransferase or sialidase, more preferably secreted serum/bloodform of the human enzyme. In a preferred embodiment the human acceptableenzyme, more preferably recombinant human acceptable enzyme, is acontrolled reagent with regard to potential harmful glycan structures,preferably NeuGc-structures according to the invention. The recombinantproteins may contain harmful glycosylation structures and inventorsrevealed that these kinds of structures are also present on recombinantglycosyltransferases, even on secreted (truncated) recombinantglycosyltransferases.

mRNA Corresponding to Glycosvlation Enzymes

The present invention is further directed to correlation of specificmessenger mRNA molecules with the preferred glycan structures accordingto the present invention. It is realized that glycosylation can becontrolled in multiple levels and one of them is transcription. Thepresence of glycosylated structures may in some case correlate withmRNAs involved in the synthesis of the structures.

The present invention is especially directed to analysis of mRNA-specieshaving correlation with expressed fucosylated glycan structures and“terminal HexNAc” containing structures preferred according to thepresent invention. The preferred mRNA-species includes mRNAcorresponding to fucosyltransferases andN-acetylglucosaminyltransferases.

Observation of Glycan Binding Structures, Lectins, CorrespondingmRNA-markers

The invention further revealed changes in mRNA-expression ofglycosylation recognizing lectins such as galectins. The cells werefurther revealed to contain lactosamine receptors for lectins. Theinvention is especially directed to analysis of expression levels ofhuman lectins and lactosamine galectin receptors, preferably analysis ofgalectins and selectins more preferably galectins for analysis of statusof cells according to the present invention.

The invention specifically revealed novel NeuGc(N-glycolylneuraminicacid)-binding lectin activity from human embryonal stem cells. Thelectin lectin recognizes polyvalent NeuGc but does not bind effectivelyto polyvalent NeuNAc. The present invention is especially directed tolabelling cells according to the invention by carbohydrate conjugatesbinding cells according to the invention, preferably labelled conjugatesof NeuGc. The invention is further directed to sorting and selectingcells, and cell derived materials and purifying proteins from cells,using labelled carbohydrate conjugates, pereferably, conjugates ofNeuGc.

Specific Characteristic Marker Structures and Glycome MarkerComponents/compositions

The N-glycan analysis of total profiles of released N-glycans revealedbeside the glycans above, which were verified to comprise

1) complex biantennary N-glycans, such asGalβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAcβ-,wherein the reminal N-acetyllactosamines can be elongated from Gal withNeuNAcα3 and/or NeuNAcα6 and2) terminal mannose containing N-glycans such as High-mannose glycanswith formula Hex₅₋₉HexNAc₂ and degradation products thereof comprisinglow number of mannose residues (Low mannose glycans) Hex₁₋₄HexNAc₂.

The Specific N-glycan Core Marker Structure

The glycan share common core structure according to the Formula:

[Manα3]_(n1)(Manα6)_(n2)Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAcβAsn,

wherein n1 and n2 are integers 0 or 1, independently indicating thepresence or absence of the terminal Man-residue, andwherein the non-reducing end terminal Manα3/Manα6-residues can beelongated to the complex type, especially biantennry structures or tomannose type (high-Man and/or low Man) or to hybrid type structures asdesnbed in examples.

It was further analyzed that the N-glycan compositios contained onlyvery minor amounts of glycans with additional HexNAx in comparison tomonosaccharide compositions of the complex type glycan above, whichcould indicate presence of no or very low amounts of the N-glycan corelinked GlcNAc-residues described by Stanley P M and Raju T S (JBC-(1998)273 (23) 14090-8; JBC (1996) 271 (13) 7484-93) and/or bisecting GlcNAc.The NMR-analysis further indicated that stem cell N-glycans, such as thecord blood N-glycan structures are essentially devoid of GlcNAcα6 linkedto reducing end subterminal GlcNAcβ4 of the N-glycan core. It isrealized that part of the terminal HexNAc-type structures appear torepresent bisecting GlcNAc-type type glycans, and quite low ornon-existent amounts of the GlcNAcα6-branching and also low amounts ofGlcNAcβ2-branch of Manβ4 described by Stanley and colleagues. Here,essentially devoid of indicates less than 10% of all the protein linkedN-glycans, more preferably the additional HexNAc units are prefesent inless than 8% of the stem cell N-glycans by mass spectrometric analysis.

The invention thus describes the major core structure of N-glycans inhuman stem cells verified by NMR-spectroscopy and by specificglycosidase digestions and was further quantitated to comprise acharacteristic smaller structal group glycans comprising specificterminal HexNAc group and/or bisecting GlcNAc-type structures, whichadditionally modify part of the core structure. The invention furtherreveals that the core structure is a useful target structure foranalysis of cells. The stem cells show characteristic binding withPSA-lectin, whose binding (and cytotoxic activity) is blocked byadditional GlcNAc unit blocking the recognition of the N-glycan core(Raju and Stanley JBC (1994); JBC (1996) 271 (13) 7484-93). As anexample very characteristic labelling with PSA-lectin is shown forembryonal stem cells in intracellular glycans in FIGS. 37 and 40.

The characteristic monosaccharide composition of the core structure willallow recognition of the major types of N-glycan structure groupspresent as additional modification of the core structure. Furthermorecomposition of the core structure is characteristic in massspectrometric analysis of N-glycan and allow immediate recognition forexample from Hex₁HexNAc₁-type (preferentially Man_(x)GlcNAc₁) glycansalso present in total glycome compostion.

Low-molecular Weight Glycan Marker Structures and Stem Cell GlycomeComponents

The invention describes novel low-molecular weight acidic glycancomponents within the acidic N-glycan and/or soluble glycan fractionswith characteristic monosaccharide compositions SA_(x)Hex₁₋₂HexNAc₁₋₂,wherein x indicates that the corresponding glycans are preferentiallysialylated with one or more sialic acid residues. The inventors realizedthat such glycans are novel and unusual with respect to N-glycanbiosynthesis and described mammalian cell glycan components, as revealalso by the fact that they are classified as “other (N-)glycan types” inN-glycan classification scheme of the present invention. The inventionis directed to analyzing, isolating, modifying, and/or binding to thesenovel glycan components according to the methods and uses of the presentinvention, and further to other uses of specific marker glycans asdescribed here. As demonstrated in the Examples of the presentinvention, such glycan components were specific parts of total glycomesof certain cell types and preferentially to certain stem cell types,making their analysis and use beneficial with regard to stem cells. Theinvention is further directed to stem cell glycomes and subglycomescontaining these glycan components.

Preferred Glycomes

The present invention is specifically directed to stem cell glycomes,which are essentially pure glycan mixtures comprising various glycans asdescribed in the invention preferably in proportions shown by theinvention. The essentially pure glycan mixtures comprise the key glycancomponents in proportions which are characteristics to stem cellglycomes. The preferred glycomes are obtained from human stem cellsaccording to the invention.

The invention is further directed to glycomes as products ofpurification process and variations thereof according to the invention.The products purified from stem cell materials by the simple,quantitative and effective methods according to the invention areessentially pure. The essentially pure means that the mixtures areessentially devoid of contaminations disturbing analysis by MALDI massspectrometry, preferably by MALDI-TOF mass spectrometry. The massspectra produced by the present methods from the essentially pureglycomes reveal that there is essentially no non-carbohydrate impuritieswith weight larger than trisaccharide and very low amount of lowermolecular weight impurities so that crystallization of MALDI matric ispossible and the glycan signals can be observed for broad glycomes withlarge variations of monosaccharide compositions and ranges of molecularweight as described by the invention. It is realized that thepurification of the materials from low amounts of stem cells comprisingvery broad range of cellular materials is very challenging task and thepresent invention has accomplished this.

Combination Compositions of the Preferred Glycome Mixtures with Matrixfor Analysis

Mass Spectrometric Matrix

The invention further revealed that it is possible to combine theglycomes with matrix useful for a mass spectrometric analysis and toobtain combination mixture useful for spectrometric analysis. Thepreferred mass spectrometric matrix is matrix for MALDI (matrix assistedlaser desorption ionization mass spectrometry) with mass spectrometricanalysis (abbreviated as MALDI matrix), MALDI is preferably performedwith TOF (time of flight) detection.

Preferred MALDI matrices include aromatic preferably benzene ringstructure comprising molecules with following characteristic. Thebenzene ring structure molecules preferably comprises 1-4 substituentssuch as hydroxyl, carboxylic acid or ketone groups. Known MALDI matrixeshave been reviewed in Harvey, Mass. Spec. Rev. 18, 349 (1999). Thepresent invention is especially and separately directed to specificmatrixes for analysis in negative ion mode of MALDI mass spectrometry,preferred for analysis of negatively charged (acidic, such as sialylatedand/or sulfated and/or phosphorylated) subglycome, and in positive ionmode of MALDI mass spectrometry (preferred for analysis of neutralglycomes). It is realized that the matrices can be optimized fornegative ion mode and positive ion mode.

The present invention is especially directed to glycome matrixcomposition optimized for the use in positive ion mode, and to the useof the MALDI-TOF matrix and matrix glycome composition, that isoptimized for the use in the analysis in positive ion mode, for theanalysis of glycome, preferably neutral glycome. The preferred matricesfor positive ion mode are aromatic matrices, e.g. 2,5-dihydroxybenzoicacid, 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxybenzoic acid,2,4,6-triihydroxyacetophenone or 6-aza-2-thiothymine, more preferably2,5-dihydroxybenzoic acid. The present invention is especially directedto glycome matrix composition optimized for the use in negative ionmode, and to the use of the MALDI-TOF matrix and the matrix glycomecompositions, that is optimized for the negative ion mode, for theanalysis of glycome, preferably acidic glycome. The preferred matricesfor negative ion mode are aromatic matrices, e.g.2,4,6-trihydroxyacetophenone, 3-hydroxypicolinic acid,2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoicacid/2-hydroxy-5-methoxybenzoic acid, or 6-aza-2-thiothymine, morepreferably 2,4,6-trihydroxyacetophenone. The invention is furtherdirected to analysis method and glycome-matrix compostion for theanalysis of glycome compositions, wherein the glycome compositioncomprises both negative and neutral glycome components. Preferredmatrices for analysis of negative and neutral glycome componentscomprising glycome are aromatic matrices, e.g.2,4,6-trihydroxyacetophenone, 3-hydroxypicolinic acid,2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoicacid/2-hydroxy-5-methoxybenzoic acid, or 6-aza-2-thiothymine, morepreferably 2,4,6-trihydroxyacetophenone.

The MALDI-matrix is a molecule capable of

1) Specifically and effectively co-crystallizing with glycomecomposition with the matrix, crystallizing meaning here forming a solidmixture composition allowing analysis of glycome involving two stepsbelow2) absorbing UV-light typically provided by a laser in MALDI-TOFinstrument, preferred wavelength of the light is 337 nm as defined bythe manuals of MALDI-TOF methods3) transferring energy to the glycome compostion so that these willionize and be analyzable by the MALDI-TOF mass spectrometry. The presentinvention is especially directed to compositions of glycomes in complexwith MALDI mass spectrometry matrix.

The present invention is specifically directed to methods of searchingnovel MALDI-matrixes with the above characteristic, preferably usefulfor analysis by the method below. The method for searching novelMALDI-matrixes using the method in the next paragraph.

The present invention is specifically directed to methods of analysis ofglycomes by MALDI-TOF including the steps:

1) Specifically and effectively co-crystallizing with glycomecomposition with the MALDI-TOF-matrix, crystallizing meaning hereforming a solid mixture composition allowing analysis of glycomeinvolving two steps below2) Providing UV light to crystalline sample by a laser in MALDI-TOFinstrument allowing the ionization of sample3) Analysis of the ions produced by the MALDI mass spectrometer,preferably by TOF analysis. The invention is further directed to thecombination of glycome purification methods and/or quantitative andqualitative data analysis methods according to the invention.

Crystalline Compositions of Glycomes

The present invention is further directed to essentially pure glycomecompositions in solid co-crystalline form with MALDI matrix. Theinvention is preferably a neutral and/or acidic glycome as complex witha matrix optimized for analysis of the specific glycome type, preferablyanalysis in negative ion mode with a matrix such as2,4,6-trihydroxyacetophenone. The invention is preferably a neutral (ornon-acidic) glycome as complex with a matrix optimized for analysis inpositive ion mode such as 2,5-dihydroxybenzoic acid.

The invention revealed that it is possible to analyze glycomes usingvery low amount of sample. The preferred crystalline glycome compositioncomprises between 0.1-100 pmol, more preferably 0.5-10 pmol morepreferably 0.5-5 pmol and more preferably about 0.5-3 pmol morepreferably about 0.5-2 pmol of sample co-crystallized with optimizedamount of matrix preferably about 10-200 nmol, more preferably 30-150nmol, and more preferably about 50-120 nmol and most preferably between60-90 nmols of the matrix, preferably when the matrix is2,5-dihydroxybenzoic acid. The matrix and analyte amounts are optimizedfor a round analysis spot with radius of about 1 mm and area of about0.8 mm². It is realized that the amount of materials can be changed inproportion of the area of the spot, smaller amount for smaller spot.Examples of preferred amounts per area of spot are 0.1-100 pmol/0.8 mm²and 10-200 pmol/3 mm². Preferred molar excess of matrix is about5000-1000000 fold, more preferably about 10000-500000 fold and morepreferably about 15000 to 200 000 fold and most preferably about 20000to 100000 fold excess when the matrix is 2,5-dihydroxybenzoic acid.

It is realized that the amount and relative amount of new matrix isoptimized based on forming suitable crystals and depend on chemicalstructure of the matrix. The formation of crystals is observed bymicroscope and further tested by performing test analysis by MALDI massspectrometry.

The invention is further directed to specific methods for crystallizingMALDI-matrix with glycome. Preferred method for crystallization inpositive ion mode includes steps: (1) optionally, elimination ofimpurities, like salts and detergents, which interfere with thecrystallization, (2) providing solution of glycome in H₂O or othersuitable solvent in the preferred concentration, (3) mixing the glycomewith the matrix in solution or depositing the glycome in solution on aprecrystallized matrix layer and (4) drying the solution preferably by agentle stream of air.

Preferred method for crystallization in negative ion mode includessteps: (1) optionally, elimination of impurities, like salts anddetergents, which interfere with the crystallization, (2) providingsolution of glycome in H₂O or other suitable solvent in the preferredconcentration, (3) mixing the glycome with the matrix in solution ordepositing the glycome in solution on a precrystallized matrix layer and(4) drying the solution preferably by vacuum.

Other Preferred Glycome Analysis Compostions Binder Glycome Compositions

The invention is further directed to compostions of essentially pureglycome composition with specific glycan binding molecules such aslectins, glycosidases or glycosyltransferases and other glycosylmodifying enzymes such as sulfateses and/or phosphatases and antibodies.It is realized these compostion are especially useful for analysis ofglycomes.

The present invention revealed that the complex glycome compositions canbe effectively and even quantitatively modified by glycosidases even invery low amounts. It was revealed that the numerous glycan structuressimilar to target structures of the enzymes do not prevent thedegradation by competive inhibition, especially by the enzymes used. Theinvention is specifically directed to preferred amounts directed toMALDI analysis for use in composition with a glycosylmodifying enzyme,preferably present in low amounts. Preferred enzymes suitable foranalysis include enzymes according to the examples.

The invention is further directed to binding of specific component ofglycome in solution with a specific binder. The preferred method furtherincludes affinity chromatography step for purification of the boundcomponent or analysis of the non-bound fraction and comparision it tothe glycome solution without the binding substance. Preferred bindersinclude lectins engineered to be lectins by removal of catalytic aminoacids (methods published by Roger Laine, Anomeric, Inc., USA, and ProfJukka Finne, Turku, Finland), lectins and antibodies or antibodyfragments or minimal binding domains of the proteins.

Additional Data Analysis and Related Methods

The present invention is especially directed to the use of glycome datafor production of mathematical formulas, or algorithms, for specificrecognition or identification of specific cell types or cell groups.Data analysis methods are presented e.g. in Example 23.

The invention is especially directed to selecting specific “structuralfeatures” such as mass spectrometric signals (such as indiviadual massspectrometric signal corresponding to one or several monosaccharidecompositions and/or glycan structures), or signal groups or subglycomesor signals corresponding to specific glycan classes, which arepreferably according to the invention, preferably the signal groups orgroups similar (preferably defined as specific structure group by theinvention) to ones shown in Table 41, from quantitative glycome data,preferably from quantitative glycome data according to the invention,for the analysis of status of a stem cell population. The invention isfurthermore directed to the methods of analysis of the cells by themethods involving the use of the specific signals or signal groups and amathematical algorithm for analysis of cell status.

Preferred algorithm includes use of proportion (such as %-proportion) ofthe specific signals from total signals as specific values (structuralfeatures) and creating a “glycan score”, which is algorithm showingcharacteristics/status of a cell type based on the specific proportionalsignal intensities (or quantitative presence of glycan structuresmeasured by any quantitation method such as specific binding proteins orquantitative chromatographic or electrophoresis analysis such as HPLCanalysis). Preferably signals which are, preferably most specifically,upregulated in a specific cell type(s) and signals which are, preferablymost specifically, downregulated in the cell type in comparison tocontrol cells (cell types) are selected to for the glycan score. In apreferred embodiment value(s) of downregulated signals are subtractedfrom upregulated signals when glycan score is calculated. The methodyields largest score values for a specific cell type or types selectedto be differentiated from other cell type(s).

The invention is specifically directed to methods for searchingcharacteristic structural features (values) from glycome profiling data,preferably quantitative or qualitative glycome profiling data. Thepreferred methods include methods for comparing the glycome data setsobtained from different samples, or from average data sets obtained froma group of similar samples such as paraller samples from same or similarcell preparations. Methods for searching characteristic features arebriefly described in the section: identification and classification ofdifferences in glycan datasets. The comparison of datasets of theglycome data according to the invention preferably includes calculationof relative and/or absolute differences of signals, preferably eachsignal between two data sets, and in another preferred embodimentbetween three or more datasets. The method preferably further includesstep of selecting the differing signals, or part thereof, forcalculating glycan score.

It is further realized that the analyzed glycome data has other usespreferred by the invention such as use of the selected characteristicsignals and corresponding glycan material:

1) for targets for structural analysis of glycans (preferably chemicallyby glycosidases, fragmentation mass spectrometry and/or NMR spectroscopyas shown by the present invention and/or structural analysis based onthe presence of other signals and knowledge of biosynthesis of glycans).The preferred use for targets includes estimation of chemicalcharacteristics of potential corresponding glycans for complete orpartial purification/separation of the specific glycan(s). The preferredchemical characteristics to be analysed preferably include one orseveral of following properties: a) acidity (e.g. by presence of acidicresidues such as sialic acid and/or sulfate and/or phosphate) for chargebased separation, b) molecular weight or hydrodymanamic volume affectingchromatographic separation, e.g. estimation of the elution volume in gelfiltration methods (the effect of acidic residue can be estimated fromeffects of similar structures and the “size” of HexNAc (GaNAc/GlcNAc) isin general twice the size of Hex (such as Gal, Man or Gic), c)estimation (e.g. based on composition and biosynthetic knowledge ofglycans) of presence of epitopes for specific binding reagents forlabelling identification in a mixture or for affinity purification, d)estimation of presence of target epitopes for specific glycosylmodifyingenzymes including glycosidases and/or glycosyltransferases (types ofbinding reagents) or for specific chemical modification reagents (suchas periodate for specific oxidation or acid for specific acidhydrolysis), for modification of glycans and recognition of themodification by potential chemical change such as incorporation ofradioactive label or by change of mass spectrometric signal of theglycan for labelling identification in a mixture.

2) use of the signals or partially or fully analysed glycan structurescorresponding to the signals for searching specific binding reagents forrecognition of cells which are preferably selected as described by thepresent invention (especially as described above) and in the methods foridentification and classification of differences in glycan datasetsand/or signals selected and/or tested by glycan score methods, arepreferably selected for targets for structural analysis of glycans(preferably by glycosidases, fragmentation mass spectrometry and/or NMRspectroscopy as shown by the present invention) and/or for use of thesignals or partially or fully analysed glycan structures correspondingto the signals for searching specific binding reagents for recognitionof cells.

The preferred method includes the step of comparing the values, andpreferably presenting the score values in graphs such as ones shown inFIG. 36 (example 23), and preferably evaluating the statisticsignificance of the result by a statistic analysis methods such as amathematical test for statistic significance such as Student's t-test or2-tailed Mann-Whitney U test. Cell type refers here to cells withspecific status and/or identity with possible individual variability.

It is realized that to differentiate a cell type from other(s) differentcharacteristic signals may be selected than for another cell type. Theinvention however revealed that for stem cells and especially forembryonal stem cells preferred characteristic signals include onesselected in the Examples as described above. It is realized that aglycan score can be also created with less characteristic signals orwith only part of signals and still relevant results can be obtained.The invention is further directed to methods for optimisation of glycanscore algorithms and methods for selecting signals for glycan scores.

In case the specific proportion (value) of a characteristic signal islow in comparision to other values a specific factor can be selected forincrease the relative “weight” of the value in the glycan scores to becalculated for the cell populations.

The preferred statuses of cells, to be analysed by mathematical methodssuch as algorithms using quantitative glycome profiling data accordingto the invention include differentiation status, individualcharacteristics and mutation, cell culture or storage conditions relatedstatus, effects of chemicals or biochemicals on cells, and otherstatuses described by the invention.

Stem Cell Nomenclature

The present invention is directed to analysis of all stem cell types,preferably human stem cells. A general nomenclature of the stem cells isdescribed in FIG. 44. The alternative nomenclatura of the presentinvention describe early human cells which are in a preferred embodimentequivalent of adult stem cells (including cord blood type materials) asshown in FIG. 44. Adult stem cells in bone marrow and blood isequivalent for stem cells from “blood related tissues”.

Lectins for Manipulation of Stem Cells, Especially Under Cell CultureConditions

The present invention is especially directed to use of lectins asspecific binding proteins for analysis of status of stem cells and/orfor the manipulation of stems cells.

The invention is specifically directed to manipulation of stem cellsunder cell culture conditions growing the stem cells in presence oflectins. The manipulation is preferably performed by immobilized lectinson surface of cell culture vessels. The invention is especially directedto the manipulation of the growth rate of stem cells by growing thecells in the presence of lectins, as show in Table 50.

The invention is in a preferred embodiment directed to manipulation ofstem cells by specific lectins recognizing specific glycan markerstructures according to invention from the cell surfaces. The inventionis in a preferred embodiment directed to use of Gal recognizing lectinssuch as ECA-lectin or similar human lectins such as galectins forrecognition of galectin ligand glycans identified from the cellsurfaces. It was further realized that there is specific variations ofgalectin expression in genomic levels in stem cells, especially forgalectins-1, -3, and -8. The present invention is especially directed tomethods of testing of these lectins for manipulation of growth rates ofembryonal type stem cells and for adult stem cells in bone marrow andblood and differentiating derivatives thereof.

Sorting of Stem Cells by Specific Lectins

The invention revealed use of specific lectin types recognizing cellsurface glycan epitopes according to the invention for sorting of stemcells, especially by FACS methods, most preferred cell types to besorted includes adult stem cells in blood and bone marrow, especiallycord blood cells. Preferred lectins for sorting of cord blood cellsinclude GNA, STA, GS-II, PWA, HHA, PSA, RCA, and others as shown inExample 32. The relevance of the lectins for isolating specific stemcell populations was demonstrated by double labeling with known stemcells markers, as described in Example 32.

Preferred Structures of Glycan Glycomes of Stem Cells

The present invention is especially directed to following O-glycanmarker structures of stem cells:

Core 1 type O-glycan structures following the marker compositionNeuAc₂Hex₁HexNAc₁, preferably including structures SAα3Galβ3GalNAcand/or SAα3Galβ3(Saα6)GalNAc; and Core 2 type O-glycan structuresfollowing the marker composition NeuAc₀₋₂Hex₂HexNAc₂dHex₀₋₁, morepreferentially further including the glycan seriesNeuAc₀₋₂Hex_(2+n)HexNAc_(2+n)dHex₀₋₁, wherein n is either 1, 2, or 3 andmore preferentially n is 1 or 2, and even more preferentially n is 1;more specifically preferably includingR₁Galβ4(R₃)GlcNAcβ6(R₂Galβ3)GalNAc,wherein R₁ and R₂ are independently either nothing or sialic acidresidue, preferably α2,3-linked sialic acid residue, or an elongationwith Hex_(n)HexNAc_(n), wherein n is independently an integer at least1, preferably between 1-3, most preferably between 1-2, and mostpreferably 1, and the elongation may terminate in sialic acid residue,preferably α2,3-linked sialic acid residue; andR₃ is independently either nothing or fucose residue, preferablyα1,3-linked fucose residue.

It is realized that these structures correlate with expression ofβ6GlcNAc-transferases synthesizing core 2 structures.

Preferred Branched N-acetyllactosamine Type Glycosphingolipids

The invention further revealed branched, I-type,poly-N-acetyllactosamines with two terminal Galβ4-residues fromglycolipids of human stem cells. The structures correlate withexpression of βGlcNAc-transferases capable of branchingpoly-N-acetyllactosamines and further to binding of lectins specific forbranched poly-N-acetylalctosamines. It was further noticed thatPWA-lectin had an activity in manipulation of stem cells, especially thegrowth rate thereof.

Preferred Qualitative and Quantitative Complete N-glycomes of Stem CellsHigh-mannose Type and Glucosylated N-glycans

The present invention is especially directed to glycan compositions(structures) and analysis of high-mannose type and glucosylatedN-glycans according to the formula:

Hex_(n3)HexNAc_(n4),

wherein n3 is 5, 6, 7, 8, 9, 10, 11, or 12, and n4=2.

According to the present invention, within total N-glycomes of stemcells the major high-mannose type and glucosylated N-glycan signalsinclude the compositions with 5≦n3≦10: Hex5HexNAc2 (1257), Hex6HexNAc2(1419), Hex7HexNAc2 (1581), Hex8HexNAc2 (1743), Hex9HexNAc2 (1905), andHex10HexNAc2 (2067);

and more preferably with 5≦n3≦9: Hex5HexNAc2 (1257), Hex6HexNAc2 (1419),Hex7HexNAc2 (1581), Hex8HexNAc2 (1743), and Hex9HexNAc2 (1905).

As demonstrated in the present invention by glycan structure analysis,preferably this glycan group in stem cells includes the molecularstructure (Manα)₈Manβ4GlcNAcβ4GlcNAc within the glycan signalHex9HexNAc2 (1905), and even more preferablyManα2Manα6(Manα2Manα3)Manα6(Manα2Manα2Manα3)Manβ4GlcNAcβ4GlcNAc.

Low-mannose Type N-glycans

The present invention is especially directed to glycan compositions(structures) and analysis of low-mannose type N-glycans according to theformula:

Hex_(n3)HexNAc_(n4)dHex_(n5),

wherein n3 is 1, 2, 3, or 4, n4=2, and n5 is 0 or 1.

According to the present invention, within total N-glycomes of stemcells the major low-mannose type N-glycan signals preferably include thecompositions with 2≦n3≦4: Hex2HexNAc2 (771), Hex3HexNAc2 (933),Hex4HexNAc2 (1095), Hex2HexNAc2dHex (917), Hex3HexNAc2dHex (1079), andHex4HexNAc2dHex (1241); and more preferably when n5 is 0: Hex2HexNAc2(771), Hex3HexNAc2 (933), and Hex4HexNAc2 (1095).

As demonstrated in the present invention by glycan structure analysis ofstem cells, preferably this glycan group in stem cells includes themolecular structures:

(Manα)₁₋₃Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAc within the glycan signals 771,917, 933, 1079, 1095, and 1095, andthe preferred low-Man structures includes structures common all stemcell types, tri-Man and tetra-Man structures according as indicated inTable 46 (Manα)₀₋₁Manα6(Manα3)Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAc, morepreferably the tri-Man structures:

Manα6(Manα3)Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAc

even more preferably the abundant molecular structure:Manα6(Manα3)Manβ4GlcNAcβGlcNAc within the glycan signal 933.

The invention is further directed to analysis of presence and/or absenceof structures varying characteristically between stem cells.

These include fucosylated and nonfucosylated di-Man structures,

specifically associated with certain blood associated stem cells

[Manα6]₀₋₁(Manα3)₀₋₁Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAc,

when either of the Mana-residues is present or absent.

The fucosylated structure was observed to be associated with specificblood related adult stem cells while the non-fucosylated structures wasobserved to have more varying expression in embryonal stem cells,embryoid bodies and more primitive cord blood stem cells (CD133+) and

cord blood mesenchymal cells. It is realized that the both di-Manstructures reflect have specific qualitative analytical value withregard to specific cell populations.

Quantitative Analysis Directed to the Low-Man Components

Beside the qualitative variations the low-Man glycans have specificvalue in quantitative analysis of stem cells. The present inventionrevealed that the low-Man glycans are especially useful for the analysisof status of the cells. For example the analysis in Table 38 revealedthat the amounts of the glycans vary between individual embruonal stemcells and there was changes during differentiation. The qualitativeanalysis above revealed that actually there is even more characteristicchanges of individual structures within the glycan group.

The group of low-Man glycans form a characteristic group among glycomecompositions. The relative total amount of glycans is between 5-12% inembryonal cel derived materials of Table 38. The glycan group wasrevealed also to be characteristic in other stem cells and relatedmaterials with total relative amount of glycomes of 21 to 35%, notablythe cells types, especially the more primitive LIN- and most effectivelyCD133+ cells differed clearly form the corresponding background cellpopulations, Table 5; and the two types feeder cells of the embryonalstem cells express the glycans in amounts of 7-8% of total neutralglycan glycomes, but the difference is again more pronounced withinfucosylated structures, which are rare in the feeders, Table 44. Glycomeanalysis of feeder cells is especially useful for methods fordevelopment of binder reagents for separation of feeders and stem cells.

The invention is directed to analysis of relative amounts of low-Manglycans, and to the specific quantitative glycome compositions,especially neutral glycan compositions, comprising about 1 to 40% oflow-Man glycans, more preferably between about 4 to 41% of the low-Manglycan for the analysis of stem cells according to the invention. 1 to40% of low-Man glycans and use of the composition for the analysis ofstem cells.

Fucosylated High-mannose Type N-glycans

The present invention is especially directed to glycan compositions(structures) and analysis of fucosylated high-mannose type N-glycansaccording to the formula:

Hex_(n3)HexNAc_(n4)dHex_(n5),

wherein n3 is 5, 6, 7, 8, or 9, n4=2, and n5=1.

According to the present invention, within total N-glycomes of stemcells the major fucosylated high-mannose type N-glycan signalpreferentially is the composition Hex5HexNAc2dHex (1403). Asdemonstrated in the present invention by glycan structure analysis ofstem cells, more preferably this glycan signal in stem cells includesthe molecular structure (Manα)₄Manβ4GlcNAcβ4(Fucα6)GlcNAc.

Soluble Glycans

The present invention is especially directed to glycan compositions(structures) and analysis of neutral soluble N-glycan type glycansaccording to the formula:

Hex_(n3)HexNAc_(n4),

wherein n3 is 1, 2, 3, 4, 5, 6, 7, 8, or 9, andn4=1.

Within total N-glycomes of stem cells the major high-mannose type andglucosylated N-glycan signals include the compositions with 4≦n3≦8, morepreferably 4≦n3≦7: Hex4HexNAc (892), Hex5HexNAc (1054), Hex6HexNAc(1216), Hex7HexNAc (1378). In the most preferred embodiment of thepresent invention, the major glycan signal in this group within totalN-glycomes of stem cells is Hex5HexNAc (1054).

The inventors were able to determine the molecular structures of thisglycan group with a combination of mass spectrometry, exoglycosidasedigestions, and nuclear magnetic resonance spectroscopy. Therefore, inanother embodiment of the present invention, preferably this glycangroup in stem cells includes the N-glycan type molecular structuresHex_(h)[(Manα3)Manβ4GlcNAc], wherein h=n3−2, even more preferably whenHex are Manα.

Neutral Monoantennary or Hybrid-type N-glycans

The present invention is especially directed to glycan compositions(structures) and analysis of neutral monoantennary or hybrid-typeN-glycans according to the formula:

Hex_(n3)HexNAc_(n4)dHex_(n5),

wherein n3 is an integer greater or equal to 2, n4=3, and n5 is aninteger greater or equal to 0.

According to the present invention, within total N-glycomes of stemcells the major neutral monoantennary or hybrid-type N-glycan signalspreferentially include the compositions with 2≦n3≦8 and 0≦n5≦2, morepreferentially compositions with 3≦n3≦6 and 0≦n5≦1, with the provisothat when n3=6 also n5=0: Hex3HexNAc3 (1136), Hex3HexNAc3dHex (1282),Hex4HexNAc3 (1298), Hex4HexNAc3dHex (1444), Hex5HexNAc3 (1460),Hex5HexNAc3dHex (1606), and Hex6HexNAc3 (1622).

According to the present invention, the total N-glycomes of culturedhuman BM MSC, CB MSC, and cells differentiated from them preferentiallyadditionally include the following structures: Hex2HexNAc3dHex (1120),Hex4HexNAc3dHex2 (1590), Hex5HexNAc3dHex2 (1752), Hex6HexNAc3dHex(1768), and Hex7HexNAc3 (1784).

In a preferred embodiment of the present invention, the N-glycan signalHex5HexNAc3 (1460), more preferentially also Hex6HexNAc3 (1622), andeven more preferentially also Hex5HexNAc3dHex (1606), containnon-reducing terminal Manα.

Neutral Complex-type N-glycans

The present invention is especially directed to glycan compositions(structures) and analysis of neutral complex-type N-glycans according tothe formula:

Hex_(n3)HexNAc_(n4)dHex_(n5),

wherein n3 is an integer greater or equal to 3, n4 is an integer greateror equal to 4, and n5 is an integer greater or equal to 0.

Within the total N-glycomes of stem cells the major neutral complex-typeN-glycan signals preferentially include the compositions with 3≦n3≦8,4≦n4≦7, and 0≦n5≦4, more preferentially the compositions with 3≦n3≦5,n4=4, and 0≦n5≦1, with the proviso that when n3 is 3 or 4, then n5=1:Hex3HexNAc4dHex (1485), Hex4HexNAc4dHex (1647), Hex5HexNAc4 (1663),Hex5HexNAc4dHex (1809); and even more preferentially also including thecomposition Hex3HexNAc5dHex (1688).

In another embodiment of the present invention, the total N-glycomes ofcultured human BM MSC, CB MSC, and cells differentiated from thempreferentially include in the major neutral complex-type N-glycansignals the compositions with 3≦n3≦5, n3=4, and 0≦n5≦1, as well as thecompositions with 5≦n4≦6, n3=n4+1, and 0≦n5≦1,

and even more preferentially also including the compositionHex3HexNAcSdHex: Hex3HexNAc4 (1339), Hex3HexNAc4dHex (1485), Hex4HexNAc4(1501), Hex4HexNAc4dHex (1647), Hex5HexNAc4 (1663), Hex5HexNAc4dHex(1809), Hex6HexNAc5 (2028), Hex6HexNAc5dHex (2174), Hex7HexNAc6 (2393),Hex7HexNAc6dHex (2539), and Hex3HexNAc5dHex (1688).

In another embodiment of the present invention, the total N-glycomes ofcultured hESC and cells differentiated from them preferentially furtherinclude in the major neutral complex-type N-glycan signalHex4HexNAc5dHex (1850).

In another embodiment of the present invention, the N-glycan signalHex3HexNAc4dHex (1485) contains non-reducing terminal GlcNAcβ, and morepreferentially the total N-glycome includes the structure:GlcNAcβ2Manα3(GlcNAcβ2Manα6)Manβ4GlcNAcβ4(Fucα6)GlcNAc (1485).

In yet another embodiment of the present invention, within the totalN-glycome of stem cells, the N-glycan signal Hex5HexNAc4dHex (1809),more preferentially also Hex5HexNAc4 (1663), contain non-reducingterminal β1,4-Gal. Even more preferentially the total N-glycome includesthe structure: Galβ4GlcNAβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAc(1663); and in a further preferred embodiment the total N-glycomeincludes the structure:Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4(Fucα6)GlcNAc (1809).

Neutral Fucosylated N-glycans

The present invention is especially directed to glycan compositions(structures) and analysis of neutral fucosylated N-glycans according tothe formula:

Hex_(n3)HexNAc_(n4)dHex_(n5),

wherein n5 is an integer greater than or equal to 1.

Within the total N-glycomes of stem cells the major neutral fucosylatedN-glycan signals preferentially include glycan compositions wherein1≦n5≦4, more preferentially 1≦n5≦3, even more preferentially 1≦n5≦2, andfurther more preferentially compositions Hex3HexNAc2dHex (1079), morepreferentially also Hex2HexNAc2dHex (917), and even more preferentiallyalso Hex5HexNAc4dHex (1809).

The inventors further found that within the total N-glycomes of stemcells a major fucosylation form is N-glycan core α1,6-fucosylation. In apreferred embodiment of the present invention, major fucosylatedN-glycan signals contain GlcNAcβ4(Fucα6)GlcNAc reducing end sequence.

The inventors further found that stem cell total N-glycomes containα1,2-Fuc, α1,3-Fuc, and/or α1,4-Fuc epitopes in a differentiation stagedependent manner. In a preferred embodiment of the present invention,major fucosylated N-glycan signals of stem cells contain α1,2-Fuc,α1,3-Fuc, and/or α1,4-Fuc epitopes, more preferentially inmultifucosylated N-glycans, wherein 2≦n5≦4.

Within the total N-glycomes of BM and CB MSC the major neutralmultifucosylated N-glycan signals preferentially include the compositionHex5HexNAc4dHex2 (1955), more preferentially also Hex5HexNAc4dHex3(2101), even more preferentially also Hex4HexNAc3dHex2 (1590), andfurther more preferentially also Hex6HexNAc5dHex2 (2320).

Within the total N-glycomes of hESC the major neutral multifucosylatedN-glycan signals preferentially include the composition Hex5HexNAc4dHex2(1955), more preferentially also Hex5HexNAc4dHex3 (2101), even morepreferentially also Hex4HexNAc5dHex2 (1996), and further morepreferentially also Hex4HexNAc5dHex3 (2142).

Neutral N-glycans with Non-reducing Terminal HexNAc

The present invention is especially directed to glycan compositions(structures) and analysis of neutral N-glycans with non-reducingterminal HexNAc according to the formula:

Hex_(n3)HexNAc_(n4)dHex,

wherein n4≧n3.

Preferably these glycan signals include Hex3HexNAc4dHex (1485) in allstem cell types; additionally preferably including Hex3HexNAc4 (1339),Hex3HexNAc4 (1339), and/or Hex3HexNAc5 (1542) in CB and BM MSC as wellas cells differentiated directly from them; additionally preferablyincluding Hex4HexNAx5 (1704), Hex4HexNAc5dHex (1850), and/orHex4HexNAc5dHex2 (1996) in hESC and cells differentiated directly fromthem; additionally preferably including Hex5HexNAc5 (1866) and/orHex5HexNAc5dHex (2012) in EB and st.3 differentiated cells (from hESC),as well as adipocyte and osteoblast differentiated cells (firom CB MSCand BM MSC, respectively).

Acidic Hybrid-type or Monoantennary N-glycans

The present invention is especially directed to glycan compositions(structures) and analysis of acidic hybrid-type or monoantennaryN-glycans according to the formula:

NeuAc_(n1)NeuGc_(n2)Hex_(n3)HexNAc_(n4)dHex_(n5)SP_(n6),

wherein n1 and n2 are either independently 1, 2, or 3; n3 is an integerbetween 3-9; n4 is 3; n5 is an integer between 0-3; and n6 is an integerbetween 0-2; with the proviso that the sum n1+n2+n6 is at least 1.

Within the total N-glycomes of stem cells the major acidic hybrid-typeor monoantennary N-glycan signals preferentially include glycancompositions wherein 3≦n3≦6, more preferentially 3≦n5≦5, and furthermore preferentially compositions NeuAcHex4HexNAc3dHex (1711),preferentially also NeuAcHex5HexNAc3dHex (1873).

Acidic Complex-type N-glycans

The present invention is especially directed to glycan compositions(structures) and analysis of acidic complex-type N-glycans according tothe formula:

NeuAc_(n1)NeuGc_(n2)Hex_(n3)HexNAc_(n4)dHex_(n5)SP_(n6),

wherein n1 and n2 are either independently 1, 2, 3, or 4; n3 is aninteger between 3-10; n4 is an integer between 4-9; n5 is an integerbetween 0-5; and n6 is an integer between 0-2; with the proviso that thesum n1+n2+n6 is at least 1.

Within the total N-glycomes of stem cells the major acidic complex-typeN-glycan signals preferentially include glycan compositions wherein4≦n4≦8, more preferentially 4≦n4≦6, more preferentially 4≦n4≦5 andfurther more preferentially compositions NeuAcHex5HexNAc4 (1930),NeuAcHex5HexNAc4dHex (2076), NeuAc2Hex5HexNAc4 (2221),NeuAcHex5HexNAc4dHex2 (2222), and NeuAc2Hex5HexNAc4dHex (2367); furthermore preferentially also NeuAc2Hex6HexNAc5dHex (2732), and morepreferentially also NeuAcHex5HexNAc5dHex (2279);

and in BM and CB MSC as well as cells directly differentiated from them,further more preferentially also NeuAc2Hex6HexNAc5 (2586) and morepreferentially also NeuAc2Hex7HexNAc6 (2952).

Modified Glycan Types

The inventors found that stem cell total N-glycomes; andsoluble+N-glycomes further contain characteristic modified glycansignals, including sialylated fucosylated N-glycans, multifucosylatedglycans, sialylated N-glycans with terminal HexNAc (the N>H and N═Hsubclasses), and sulphated or phosphorylated N-glycans, which aresubclasses of the abovementioned glycan classes. According to thepresent invention, their quantitative proportions in different stem celltypes have characteristic values as described in Table 51.

Phosphorylated and Sulphated Glycans

Specifically, major phosphorylated glycans typical to stem cells includeHex5HexNAc2(HPO₃) (1313), Hex6HexNAc2(HPO₃) (1475), andHex7HexNAc2(HPO₃) (1637);

and major sulphated glycans typical to stem cells includeHex5HexNAc4dHex(SO₃) (1865) and more preferentially alsoHex6HexNAc3(SO₃) (1678).

According to the present invention, their quantitative proportions indifferent stem cell types preferentially have characteristic values asdescribed in Table 51.

Preferred Combinations of Glycan Types in Complete Glycomes

The preferred complete glycomes of stem cells include glycan types ofthe four following types: 1) high-mannose type, 2) low-mannose type, 3)hybrid-type or monoantennary, and 3) complex-type N-glycans,

which more preferentially contain fucosylated glycans, even morepreferentially also sialylated glycans, and further more preferentiallyalso sulphated and/or phosphorylated glycans;and most preferentially also including soluble glycans as described inthe present invention.

In a preferred embodiment of the preferred glycan type combinationswithin the stem cell complete glycomes, their relative abundances are asdescribed in Table 51.

Preferred Binders for Stem Cell Sorting and Isolation

As described in the Examples, the inventors found that especially themannose-specific and especially α1,3-linked mannose-binding lectin GNAwas suitable for negative selection enrichment of CD34+ stem cells fromCB MNC. In addition, the poly-LacNAc specific lectin STA and thefucose-specific and especially α1,2-linked fucose-specific lectin UEAwere suitable for positive selection enrichment of CD34+ stem cells fromCB MNC.

The present invention is specifically directed to stem cell bindingreagents, preferentially proteins, preferentially mannose-binding orα1,3-linked rnannose-binding, poly-LacNAc binding, LacNAc-binding,and/or fucose- or preferentially α1,2-linked fucose-binding; in apreferred embodiment stem cell binding or nonbinding lectins, morepreferentially GNA, STA, and/or UEA; and in a further preferredembodiment combinations thereof; to uses described in the presentinvention taking advantage of glycan-binding reagents that selectivelyeither bind to or do not bind to stem cells.

Preferred Uses for Stem Cell Type Specific Galectins and/or GalectinLigands

As described in the Examples, the inventors also found that differentstem cells have distinct galectin expression profiles and also distinctgalectin (glycan) ligand expression profiles. The present invention isfurther directed to using galactose-binding reagents, preferentiallygalactose-binding lectins, more preferentially specific galectins; in astem cell type specific fashion to modulate or bind to certain stemcells as described in the present invention to the uses described. In afurther preferred embodiment, the present invention is directed to usinggalectin ligand structures, derivatives thereof, or ligand-mimickingreagents to uses described in the present invention in stem cell typespecific fashion.

EXAMPLES Example 1 Glycan Isolation and Analysis Examples of GlycanIsolation Methods

Glycan isolation. N-linked glycans are preferentially detached fromcellular glycoproteins by F. meningosepticum N-glycosidase F digestion(Calbiochem, USA) essentially as described previously (Nyman et al.,1998), after which the released glycans are preferentially purified foranalysis by solid-phase extraction methods, including ion exchangeseparation, and divided into sialylated and non-sialylated fractions.For O-glycan analysis, glycoproteins are preferentially subjected toreducing alkaline O-elimination essentially as described previously(Nyman et al., 1998), after which sialylated and neutral glycan alditolfractions are isolated as described above. Free glycans arepreferentially isolated by extracting them from the sample with water.

Example of a glycan purification method. Isolated oligosaccharides canbe purified from complex biological matrices as follows, for example forMALDI-TOF mass spectrometric analysis. Optionally, contaminations areremoved by precipitating glycans with 80-90% (v/v) aqueous acetone at−20° C., after which the glycans are extracted from the precipitate with60% (v/v) ice-cold methanol. After glycan isolation, the glycanpreparate is passed in water through a strong cation-exchange resin, andthen through C₁₈ silica resin. The glycan preparate can be furtherpurified by subjecting it to chromatography on graphitized carbonmaterial, such as porous graphitized carbon (Davies, 1992). To increasepurification efficiency, the column can be washed with aqueoussolutions. Neutral glycans can be washed from the column and separatedfrom sialylated glycans by elution with aqueous organic solvent, such as25% (v/v) acetonitrile. Sialylated glycans can be eluted from the columnby elution with aqueous organic solvent with added acid, such as 0.05%(v/v) trifluoroacetic acid in 25% (v/v) acetonitrile, which elutes bothneutral and sialylated glycans. A glycan preparation containingsialylated glycans can be further purified by subjecting it tochromatography on microcrystalline cellulose in n-butanol:ethanol:water(10:1:2, v/v) and eluted by aqueous solvent, preferentially 50%ethanol:water (v/v). Preferentially, glycans isolated from small sampleamounts are purified on miniaturized chromatography columns and smallelution and handling volumes. An efficient purification method comprisesmost of the abovementioned purification steps. In an efficientpurification sequence, neutral glycan fractions from small samples arepurified with methods including carbon chromatography and separateelution of the neutral glycan fraction, and glycan fractions containingsialylated glycans are purified with methods including both carbonchromatography and cellulose chromatography.

MALDI-TOF mass spectrometry. MALDI-TOF mass spectrometry is performedwith a Voyager-DE STR BioSpectrometry Workstation or a Bruker UltraflexTOF/TOF instrument, essentially as described previously (Saarinen etal., 1999; Harvey et al., 1993). Relative molar abundancies of bothneutral (Naven & Harvey, 1996) and sialylated (Papac et al., 1996)glycan components are assigned based on their relative signalintensities. The mass spectrometric fragmentation analysis is done withthe Bruker Ultraflex TOF/TOF instrument according to manufacturer'sinstructions.

Results

Examples of analysis sensitivity. Protein-linked and free glycans,including N- and O-glycans, are typically isolated from as little asabout 5×10⁴ cells in their natual biological matrix and analyzed byMALDI-TOF mass spectrometry.

Examples of analysis reproducibility and accuracy. The present glycananalysis methods have been validated for example by subjecting a singlebiological sample, containing human cells in their natural biologicalmatrix, to analysis by five different laboratory personnel. The resultswere highly comparable, especially by the terms of detection ofindividual glycan signals and their relative signal intensities,indicating that the reliability of the present methods in accuratelydescribing glycan profiles of biological samples including cells isexcellent. Each glycan isolation and purification phase has beencontrolled by its reproducibility and found to be very reproducible. Themass spectrometric analysis method has been validated by syntheticoligosaccharide mixtures to reproduce their molar proportions in amanner suitable for analysis of complex glycan mixtures and especiallyfor accurate comparison of glycan profiles from two or more samples. Theanalysis method has also been successfully transferred from one massspectrometer to another and found to reproduce the analysis results fromcomplex glycan profiles accurately by means of calibration of theanalysis.

Examples of biological samples and matrices for successfid glycananalysis. The method has been successfully implied on analysis of e.g.blood cells, cell membranes, aldehyde-fixated cells, glycans isolatedfrom glycolipids and glycoproteins, free cellular glycans, and freeglycans present in biological matrices such as blood. The experienceindicates that the method is especially useful for analysis ofoligosaccharide and similar molecule mixtures and their optional andoptimal purification into suitable form for analysis.

Example 2 Glycan Profiling

Generation of glycan profiles from mass spectrometric data FIG. 1A showsa MALDI-TOF mass spectrum recorded in positive ion mode from a sample ofneutral N-glycans. The profile includes multiple signals that interferewith the interpretation of the original sample's glycosylation,including non-glycan signals and multiple signals arising from singleglycan signals. According to the present invention, the massspectrometric data is transformed into a glycan profile (FIG. 1B), whichrepresents better the original glycan profile of the sample. Anexemplary procedure is briefly as follows, and it includes followingsteps: 1) The mass spectrometric signals are first assigned to proposedmonosaccharide compositions e.g. according to Table 1. 2) The massspectrometric signals of ions in the molecular weight are of glycansignals typically show isotopic patterns, which can be calculated basedon natural abundancies of the isotopes of the elements in the Earth'scrust. The relative signal intensities of mass spectrometric signalsnear each other can be overestimated or underestimated, if theirisotopic patterns are not taken into account. According to the presentmethod, the isotopic patterns are calculated for glycan signals neareach other, and relative intensities of glycan signals corrected basedon the calculations. 3) Glycan ions are predominantly present as [M+Na]+ions in positive ion mode, but also as other adduct ions such as [M+K]+.The proportion of relative signal intensities of [M+Na]+ to [M+K]+ ionsis deduced from several signals in the spectrum, and the proportion isused to remove the effect of [M+K]+ adduct ions from the spectrum. 4)Other contaminating mass spectrometric signals not arising from theoriginal glycans in the sample can optionally be removed from theprofile, such as known contaminants, products of elimination of water,or in a case of permethylated oligosaccharides, undermethylated glycansignals. 5) The resulting glycan signals in the profile are normalized,for example to 100%, for allowing comparison between samples.

FIG. 2A shows a MALDI-TOF mass spectrum recorded in negative ion modefrom a sample of neutral N-glycans. The profile includes multiplesignals that interfere with the interpretation of the original sample'sglycosylation, including non-glycan signals and multiple signals arisingfrom single glycan signals. According to the present invention, the massspectrometric data is transformed into a glycan profile (FIG. 2B), whichrepresents better the original glycan profile of the sample. Anexemplary procedure is briefly as follows, and it includes followingsteps: 1) The mass spectrometric signals are first assigned to proposedmonosaccharide compositions e.g. according to Table 2. 2) The massspectrometric signals of ions in the molecular weight are of glycansignals typically show isotopic patterns, which can be calculated basedon natural abundancies of the isotopes of the elements in the Earth'scrust. The relative signal intensities of mass spectrometric signalsnear each other can be overestimated or underestimated, if theirisotopic patterns are not taken into account. According to the presentmethod, the isotopic patterns are calculated for glycan signals neareach other, and relative intensities of glycan signals corrected basedon the calculations. 3) Glycan ions are predominantly present as [M-H]−ions in negative ion mode, but also as ions such as [M-2H+Na]− or [M-2H+K]−. The proportion of relative signal intensities of e.g. [M-H]− to[M-2H+Na]− and [M-2H+ K]− ions is deduced from several signals in thespectrum, and the proportion is used to remove the effect of e.g. theseadduct ions from the specs 4) Other contaminating mass spectrometricsignals not arising from the original glycans in the sample canoptionally be removed from the profile, such as known contaminants orproducts of elimination of water. 5) The resulting glycan signals in theprofile are normalized, for example to 100%, for allowing comparisonbetween samples.

Example 3 MALDI-TOF Mass Spectrometric N-glycan Profiling of Cord BloodMononuclear Cell Populations and Peripheral Blood Mononuclear CellsExamples of Cell Material Production Cord Blood Cell Populations

Preparation of mononuclear cells. Cord blood was diluted 1:4 withphosphate buffered saline (PBS)-2 mM EDTA and 35 ml of diluted cordblood was carefully layered over 15 ml of Ficoll-Paque® (AmershamBiociences, Piscataway, USA). Tubes were centrifuged for 40 minutes at400 g without brake. Mononuclear cell layer at the interphase wascollected and washed twice in PBS-2 mM EDTA. Tubes were centrifuged for10 minutes at 300 g.

Positive selection of CD34+/CD133+ cells. The cord blood mononuclearcell pellet was resuspended in a final volume of 300 μl of PBS-2 mMEDTA-0.5% BSA (Sigma, USA) per 10⁸ total cells. To positively selectCD34+ or CD133+ cells, 100 μl of FcR Blocking Reagent and 100 μl CD34 orCD133 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) wereadded per 10⁸ mononuclear cells cells. Suspension was incubated for 30minutes at 6-12° C. Cells were washed with PBS-2 mM EDTA-0.5% BSA andresuspended in 500 μl of PBS-2 mM EDTA-0.5% BSA per 10⁸ cells.

The appropriate MACS affinity column type (Miltenyi Biotec, BergischGladbach, Germany) was chosen according to the number of total cells: MScolumn for <2×10⁸ cells and LS column for 2×10⁸-2×10⁹ cells. The columnwas placed in the magnetic field and rinsed with PBS-2 mM EDTA-0.5% BSA.Labeled cell suspension was applied to the column and the cells passingthrough the column were collected as the negative cell fraction (CD34−or CD133−). The column was then washed four times with PBS-2mM EDTA-0.5%BSA. The column was removed from the magnetic field and the retainedpositive cells (CD34+ or CD133⁺) were eluted with PBS-2 mM EDTA-0.5% BSAusing a plunger.

The eluted positive cells were centrifuged for 5 minutes at 300 g andresuspended in 300 μl PBS-2 mM EDTA-0.5% BSA. 25 μl of FcR BlockingReagent and 25 μl CD34 or CD133 Microbeads were added. Suspension wasincubated for 15 minutes at 6-12° C. Cells were washed with PBS-2 mMEDTA-0.5% BSA and resuspended in 500 μl of PBS-2 mM EDTA-0.5% BSA.

A MS column was placed in the magnetic field and rinsed with PBS-2mMEDTA-0.5% BSA. Labeled cell suspension was applied to the column. Thecolumn was washed four times with PBS-2 mM EDTA-0.5% BSA. The column wasthen removed from the magnetic field and the retained positive cells(CD34+ or CD133+) were eluted with PBS-2 mM EDTA-0.5% BSA using aplunger.

Negative selection of Lin− cells. To deplete lineage committed cells,mononuclear cells (8×10⁷/ml) in PBS-0.5% BSA were labeled with 100 μl/mlcells with StemSep Progenitor Enrichment Cocktail containing antibodiesagainst CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, Glycophorin A(StemCell Technologies, Vancouver, Canada) at room temperature for 15minutes. Subsequently, 60 μl of colloidal magnetic iron particles wereadded per 1 ml cell suspension and incubated at room temperature for 15minutes.

The labeled cell suspension was loaded into MACS LD column (MiltenyiBiotec) and unlabeled cells passing through the column were collected asthe negative fracction (Lin−). LD column was washed twice with 1 mlPBS-0.5% BSA and effluents were collected into the same tube withunlabelled cells. The column was then removed from the magnetic fieldand the retained positive cells (Lin+) were eluted with PBS-0.5% BSAusing a plunger.

Results

Glycan isolation from mononuclear cell populations. Mononuclear cellswere isolated from one sample of peripheral blood, as well as cord bloodsamples from multiple donors. The cord blood mononuclear cells werefurther affinity-purified into CD34+, CD34−, CD133+, CD133−, Lin+, andLin− cell samples, as described under Experimental procedures. N-glycanswere isolated from the samples, and glycan profiles were generated fromMALDI-TOF mass spectrometry data of isolated neutral and sialylatedN-glycan fractions as described in the preceding examples.

Neutral N-glycan profiles. Neutral N-glycan profiles obtained from cordblood and peripheral blood mononuclear cells are presented in Table 3.The present results from cord blood cell populations are averaged frommultiple experiments and multiple cord blood donors, while theperipheral blood cell results are exemplary results obtained from asingle experiment. From the present results, it is evident that cordblood cell populations differ from each other and from peripheral bloodcells with respect to their neutral N-glycan profiles. Differences inthe glycan profiles between cell populations were consistent throughoutmultiple samples and experiments, and multiple individual glycan signalshad consistently differing relative abundancies. The analysis revealedin each cell type the relative proportions of about 25-55 glycan signalsthat were assigned as non-sialylated N-glycan components.

Neutral N-glycan structural features. Neutral N-glycan groupingsproposed for cord blood cell populations, cord blood mononuclear cells(CB MNC), and peripheral blood mononuclear cells (PB MNC) are presentedin Table 5. In comparison of cord blood stem cell populations (CD34+,CD133+, and Lin−) and the corresponding stem cell depleted cord bloodmononuclear cells, numerous cell-type specific features could beidentified

Identification of soluble glycan components. In the present analysis,neutral glycan components were identified in all the cell types thatwere assigned as soluble glycans based on their proposed monosaccharidecompositions Hex₂₋₉HexNAc₁ and Hex₁₂HexNAc₁, and these glycan signalshave been omitted from Table 3. The abundancies of these glycancomponents in relation to each other and in relation to the other glycansignals varied between individual samples and cell types. Indicationsfor the presence of such glycans have previously been described incertain human cells (Moore, 1999). The relative proportions ofHex₂₋₉HexNAc₁ and Hex₁₂HexNAc₁ glycan signals are typically reduced ifglycoprotein fractions are isolated from cord blood cell populations andwashed, indicating that these glycan components are present in thesoluble fraction of cells and not covalently bound to glycoproteins.

Sialylated N-glycan profiles. Sialylated N-glycan profiles obtained fromcord blood and peripheral blood mononuclear cells are presented in Table4. From the present results, it is evident that cord blood cellpopulations differ from each other and from peripheral blood cells withrespect to their sialylated N-glycan profiles. The analysis revealed ineach cell type the relative proportions of about 45-125 glycan signalsthat were assigned as acidic N-glycan components.

Sialylated N-glycan structural features. Sialylated N-glycan groupingsproposed for cord blood cell populations, cord blood mononuclear cells(CB MNC), and peripheral blood mononuclear cells (PB MNC) are presentedin Table 6. In comparison of cord blood stem cell populations (CD34+)and the corresponding stem cell depleted cord blood mononuclear cells,numerous cell-type specific features could be identified.

Conclusions

Comparison of neutral N-glycan profiles. Differences in the glycanprofiles between cell populations were consistent throughout multiplesamples and experiments, indicating that the present method of glycanprofiling and the differences in the present glycan profiles can be usedto identify the presence of certain cell types in purified human cellpopulations, or their purity. The present method and the present resultscan also be used to identify cell-type specific glycan structuralfeatures or cell-type specific glycan profiles.

Comparison of neutral N-glycan structural features. Differences inglycosylation profiles between analyzed cell types were identified basedon proposed structural features, which can be used to identify cell-typespecific glycan structural features. Identified cell-type specificfeatures of neutral N-glycan profiles are concluded below:

CD34+:

-   1) Lower amounts of larger neutral N-glycans.

CD133+:

-   1) Lower amounts of larger neutral N-glycans;-   2) Lower amounts of neutral N-glycans containing two or more    deoxyhexose residues per chain, indicating reduced expression of    neutral N-glycans containing α1,2-, α1,3-, or α1,4-linked fucose    residues;-   3) Increased amounts of terminal HexNAc residues; and-   4) Lower amounts of hybrid-type and/or monoantennary neutral    N-glycans.

Lin−:

-   1) Lower amounts of larger neutral N-glycans;-   2) Lower amounts of neutral N-glycans containing two or more    deoxyhexose residues per chain, indicating reduced expression of    neutral N-glycans containing α1,2-, α1,3-, or α1,4-linked fucose    residues; and-   3) Increased amounts of terminal HexNAc residues.

Cord blood stem cell populations in general: These neutral N-glycanprofile features were common to all of the three cell types above whencompared to corresponding stem cell depleted cord blood mononuclear cellsamples. These features are more strongly expressed in CD133+ and Lin−cell populations than in CD34+ cell population.

-   1) Lower amounts of larger neutral N-glycans;-   2) Lower amounts of neutral N-glycans containing two or more    deoxyhexose residues per chain, indicating reduced expression of    neutral N-glycans containing α1,2-, α1,3-, or α1,4-linked fucose    residues;-   3) Increased amounts of terminal HexNAc residues; and-   4) Lower amounts of low-mannose type N-glycans compared to    high-mannose type N-glycans.

Cord Blood Mononuclear Cells Compared to Peripheral Blood MononuclearCells:

-   1) Increased amounts of neutral N-glycans containing two or more    deoxyhexose residues per chain, indicating increased expression of    neutral N-glycans containing α1,2-, α1,3-, or α1,4-linked fucose    residues.

Comparison of sialylated N-glycan profiles. Differences in the glycanprofiles between cell populations were observed, indicating that thepresent method of glycan profiling and the differences in the presentglycan profiles can be used to identify the presence of certain celltypes in purified human cell populations, or their purity. The presentmethod and the present results can also be used to identify cell-typespecific glycan structural features or cell-type specific glycanprofiles.

Comparison of sialylated N-glycan structural features. Differences inglycosylation profiles between analyzed cell types were identified basedon proposed structural features, which can be used to identify cell-typespecific glycan structural features. Identified cell-type specificfeatures of sialylated N-glycan profiles are concluded below:

CD34+:

-   1) Lower amounts of larger sialylated N-glycans; and-   2) Lower amounts of potentially bisecting GlcNAc containing    sialylated N-glycans.

Example 4 MALDI-TOF Mass Spectrometric O-glycan Profiling of Cord Bloodand Peripheral Blood Mononuclear Cell Populations ExperimentalProcedures

O-glycan isolation. O-glycans were isolated from glycoproteins afterenzymatic de-N-glycosylation by N-glycosidase F and extraction ofsoluble glycans as described in the preceeding Examples. O-glycans wereliberated by reductive alkaline Pelimination essentially as described in(Nyman et al., 1998).

Results

O-glycan isolation. O-glycans were isolated from de-N-glycosylatedglycoproteins of Lin− and Lin+ cord blood mononuclear cells as describedabove, fractionated into sialylated and neutral glycan fractions, andanalyzed by MALDI-TOF mass spectrometry as described in the preceedingExamples.

O-glycan profiles. In the neutral O-glycan fraction, following O-glycansignals were detected: m/z 773, 919, 1138, and 1284, corresponding tosodium adduct ions of the O-glycan alditols Hex₂HexNAc₂,Hex₂HexNAc₂dHex₁, Hex₃HexNAc₃, and Hex₂HexNAc₂dHex₁, respectively. Therelative amounts of the signals differed between cell types. In Lin−cells, the relationship of the amounts of Hex₂HexNAc₂ andHex₂HexNAc₂dHex₁ signals was about 2:1, which is higher than inperipheral blood mononuclear cells. In the sialylated O-glycan fraction,following O-glycan signals were detected: m/z 675, 966, 1040, 1186, and1331, corresponding to [M-H]⁻ ions of the O-glycan alditolsNeuAc₁Hex₁HexNAc₁, NeuAc₂Hex₁HexNAc₁, NeuAc₁Hex₂HexNAc₂,NeuAc₁Hex₂HexNAc₂dHex₁, and NeuAc₂Hex₂HexNAc₂, respectively. Therelative amounts of the signals differed between cell types.

Example 5 MALDI-TOF Mass Spectrometric Glycolipid Glycan Profiling ofCord Blood and Peripheral Blood Mononuclear Cell PopulationsExperimental Procedures and Results

Glycolipid and glycan isolation. Glycolipids were isolated fromperipheral blood and cord blood mononuclear cells essentially asdescribed in (Karlsson, H. et al., 2000). Sphingoglycolipids weredetached by digestion with endoglycoceramidase from Macrobdella decora(Calbiochem, USA). After the reaction, liberated glycans were purified,fractionated into sialylated and neutral glycan fractions, and analyzedby MALDI-TOF mass spectrometry as described in the preceding Examples.

Glycolipid glycan profiles. Table 7 describes the detected glycansignals and their proposed monosaccharide compositions. Relative amountsof individual signals in the profile varied between the analyzed celltypes. The monosaccharide compositions correlate with known glycolipidcore structures, such as gangliosides, lacto- and neolactoglycolipids,and globosides, and extensions of the core structures, such aspoly-N-acetyllactosamine chains. Several glycans show fucosylationand/or sialylation of the core and extended structures.

Example 6 Comparison of Freshly Isolated and Frozen-thawed cord bloodcell glycan Profiles Results

N-glycan isolation. Several CD34+, CD34−, CD133+, and CD133− cellsamples were isolated as described above from both fresh andfrozen-thawed cord blood units. N-glycans were isolated from thesamples, and glycan profiles were generated from MALDI-TOF massspectrometry data of isolated neutral and sialylated N-glycan fractionsas described in the preceding Examples.

Comparison of glycan profiles. The analysis revealed significantdifferences in the N-glycan profiles between samples that were isolatedfrom fresh cord blood units and units that were kept frozen and thawedbefore cell isolation. The differences in multiple signals in the glycanprofiles were consistent in all the analyzed samples. The majordifference in neutral N-glycan profiles was the signal at m/z 917,corresponding to Hex₂HexNAc₂dHex₁, which was the most abundant neutralN-glycan signal in the samples from frozen-thawed cord blood. Therelative abundancies of the signal groups corresponding toHex₁₋₄HexNAc₂dHex₀₋₁ and especially Hex₁₋₄HexNAc₂dHex₁ monosaccharidecompositions, were elevated in the frozen-thawed cell samples incomparison to freshly isolated cell samples.

Conclusions

According to the present results, glycan profiling can effectivelydetect changes in glycan profiles, individual glycan signals, and glycansignal groups, which are associated with differential cell treatmentconditions.

Example 7 Glycosidase Profiling of Cord Blood Mononuclear Cell N-glycansExperimental Procedures

Exoglycosidase digestions. Neutral N-glycan fractions were isolated fromcord blood mononuclear cell populations as described above.Exoglycosidase reactions were performed essentially after manufacturers'instructions and as described in (Saarinen et al., 1999). The differentreactions were; α-Man: α-mannosidase from Jack beans (C. ensiformis;Sigma, USA); β1,4-Gal: β1,4-galactosidase from S. pneumoniae(recombinant in E. coli; Calbiochem, USA); β1,3-Gal: recombinantβ1,3-galactosidase (Calbiochem, USA); GlcNAc: β-glucosaminidasefrom S.pneumoniae (Calbiochem, USA); α2,3-SA: α2,3-sialidase from S. pneumoniae(Calbiochem, USA). The analytical reactions were carefully controlledfor specificity with synthetic oligosaccharides in parallel controlreactions that were analyzed by MALDI-TOF mass spectrometry. The sialicacid linkage specificity of α2,3-SA was controlled with syntheticoligosaccharides in parallel control reactions, and it was confirmedthat in the reaction conditions the enzyme hydrolyzed α2,3-linked butnot α2,6-linked sialic acids. The analysis was performed by MALDI-TOFmass spectrometry as described in the preceding examples. Digestionresults were analyzed by comparing glycan profiles before and after thereaction.

Results

Glycosidase profiling of neutral N-glycans. Neutral N-glycan fractionsfrom affinity-purified CD34+, CD34−, CD133+, CD133−, Lin+, and Lin− cellsamples from cord blood mononuclear cells were isolated as describedabove. The glycan samples were subjected to parallel glycosidasedigestions as described under Experimental procedures. Profiling resultsare summarized in Table 8 (CD34+ and CD34− cells), Table 9 (CD133+ andCD133− cells), and Table 10 (Lin− and Lin+ cells). The present resultsshow that several neutral N-glycan signals are individually sensitivetowards all the exoglycosidases, indicating that in all the cell typesseveral neutral N-glycans contain specific substrate glycan structuresin their non-reducing termini. The results also show clear differencesbetween the cell types in both the sensitivity of individual glycansignals towards each enzyme and also profile-wide differences betweencell types, as detailed in the Tables cited above.

Glycosidase profiling ofsialylated N-glycans. Sialylated N-glycanfractions from affinity-purified CD133+ and CD133− cell samples fromcord blood mononuclear cells were isolated as described above. Theglycan samples were subjected to parallel glycosidase digestions asdescribed under Experimental procedures. Profiling results aresummarized in FIGS. 3 and 4. The results show significant differencesbetween the glycan profiles of the analyzed cell types in the sialylatedand neutral glycan fractions resulting in the reaction. The presentresults show that differences are seen in multiple signals in aprofile-wide fashion. Also individual signals differ between cell types,as discussed below.

Cord blood CD133⁺ and CD133⁻ cell N-glycans are differentiallyα2,3-sialylated. Sialylated N-glycans from cord blood CD133⁺ and CD133⁻cells were treated with α2,3-sialidase, after which the resultingglycans were divided into sialylated and non-sialylated fractions, asdescribed under Experimental procedures. Both α2,3-sialidase resistantand sensitive sialylated N-glycans were observed, i.e. after thesialidase treatment sialylated glycans were observed in the sialylatedN-glycan fraction and desialylated glycans were observed in the neutralN-glycan fraction. The results indicate that cord blood CD133⁺ andCD133⁻ cells are differentially α2,3-sialylated. For example, afterα2,3-sialidase treatment the relative proportions of monosialylated(SA₁) glycan signal at m/z 2076, corresponding to the [M-H]⁻ ion ofNeuAc₁Hex₅HexNAc₄dHex₁, and the disialylated (SA₂) glycan signal at m/z2367, corresponding to the [M-H]⁻ ion of NeuAc₂Hex₅HexNAc₄dHex₁,indicate that α2,3-sialidase resistant disialylated N-glycans arerelatively more abundant in CD133⁻ than in CD133⁺ cells, when comparedto α2,3-sialidase resistant monosialylated N-glycans (FIG. 5). It isconcluded that N-glycan α2,3-sialylation in relation to other sialicacid linkages including especially α2,6-sialylation, is more abundant incord blood CD133⁺ cells than in CD133⁻ cells.

In cord blood CD133⁻ cells, several sialylated N-glycans were observedthat were resistant to α2,3-sialidase treatment, i.e. neutral glycanswere not observed that would correspond to the desialylated forms of theoriginal sialylated glycans. The results revealing differentialα2,3-sialylation of individual N-glycan structures between cord bloodCD133⁺ and CD133⁻ cells are presented in Table 11. The present resultsindicate that N-glycan α2,3-sialylation in relation to other sialic acidlinkages is more abundant in cord blood CD133⁺ cells than in CD133⁻cells.

Sialidase analysis. The sialylated N-glycan fraction isolated from acord blood mononuclear cell population (CB MNC; FIG. 7) was digestedwith broad-range sialidase as described in the preceding Examples. Afterthe reaction, it was observed by MALDI-TOF mass spectrometry that thevast majority of the sialylated N-glycans were desialylated andtransformed into corresponding neutral N-glycans, indicating that theyhad contained sialic acid residues (NeuAc and/or NeuGc) as suggested bythe proposed monosaccharide compositions. FIG. 8 shows the glycanprofiles of combined neutral (FIG. 6) and desialylated (originallysialylated) N-glycan fractions of a CB MNC population. The profilescorrespond to total N-glycan profiles isolated from the cell samples (indesialylated form). It is calculated that approximately 25% of theN-glycan signals correspond to high-mannose type N-glycan monosaccharidecompositions, and 28% to low-mannose type N-glycans, 34% to complex-typeN-glycans, and 13% to hybrid-type or monoantennary N-glycansmonosaccharide compositions.

Conclusions

The present results suggest that 1) the glycosidase profiling method canbe used to analyze structural features of individual glycan signals, aswell as differences in individual glycans between cell types, 2)different cell types differ from each other with respect to bothindividual glycan signals' and glycan profiles' susceptibility toglycosidases, and 3) glycosidase profiling can be used as a furthermeans to distinguish different cell types, and in such case theparameters for comparison are both individual signals and profile-widedifferences.

Example 8 MALDI-TOF Mass Spectrometric N-glycan Profiling and LectinProfiling of Cord Blood Derived and Bone Marrow Derived Mesenchymal StemCell Lines Examples of Cell Sample Production Cord Blood DerivedMesenchymal Stem Cell Lines

Collection of umbilical cord blood. Human term umbilical cord blood(UCB) units were collected after delivery with informed consent of themothers and the UCB was processed within 24 hours of the collection. Themononuclear cells (MNCs) were isolated from each UCB unit diluting theUCB 1:1 with phosphate-buffered saline (PBS) followed by Ficoll-PaquePlus (Amersham Biosciences, Uppsala, Sweden) density gradientcentrifugation (400 g/40 min). The mononuclear cell fragment wascollected from the gradient and washed twice with PBS.

Umbilical cord blood cell isolation and culture. CD45/Glycophorin A(GlyA) negative cell selection was performed using immunolabeledmagnetic beads (Miltenyi Biotec). MNCs were incubated simultaneouslywith both CD45 and GlyA magnetic microbeads for 30 minutes andnegatively selected using LD columns following the manufacturer'sinstructions (Miltenyi Biotec). Both CD45/GlyA negative elution fractionand positive fraction were collected, suspended in culture media andcounted CD45/GlyA positive cells were plated on fibronectin (FN) coatedsix-well plates at the density of 1×10⁶/cm². CD45/GlyA negative cellswere plated on FN coated 96-well plates (Nunc) about 1×10⁴ cells/well.Most of the non-adherent cells were removed as the medium was replacednext day. The rest of the non-adherent cells were removed duringsubsequent twice weekly medium replacements.

The cells were initially cultured in media consisting of 56% DMEM lowglucose (DMEM-LG, Gibco, http://www.invitrogen.com) 40% MCDB-201(Sigma-Aldrich) 2% fetal calf serum (FCS), 1× penicillin-streptomycin(both form Gibco), 1×ITS liquid media supplement(insulin-transfer-selenium), 1× linoleic acid-BSA, 5×10⁻⁸ Mdexamethasone, 0.1 mM L-ascorbic acid-2-phosphate (all three fromSigma-Aldrich), 10 nM PDGF (R&D systems, http://www.RnDSystems.com) and10 nM EGF (Sigma-Aldrich). In later passages (after passage 7) the cellswere also cultured in the same proliferation medium except the FCSconcentration was increased to 10%.

Plates were screened for colonies and when the cells in the colonieswere 80-90% confluent the cells were subcultured. At the first passageswhen the cell number was still low the cells were detached with minimalamount of trypsin/EDTA (0.25%/1 mM, Gibco) at room temperature andtrypsin was inhibited with FCS. Cells were flushed with serum freeculture medium and suspended in normal culture medium adjusting theserum concentration to 2%. The cells were plated about 2000-3000/cm². Inlater passages the cells were detached with trypsin/EDTA from definedarea at defined time points, counted with hematocytometer and replatedat density of 2000-3000 cells/cm².

Bone Marrow Derived Mesenchymal Stem Cell Lines

Isolation and culture of bone marrow derived stem cells. Bone marrow(BM)-derived MSCs were obtained as described by Leskelä et al. (2003).Briefly, bone marrow obtained during orthopedic surgery was cultured inMinimum Essential Alpha-Medium (α-MEM), supplemented with 20 mM HEPES,10% FCS, 1× penicillin-streptomycin and 2 mM L-glutamine (all fromGibco). After a cell attachment period of 2 days the cells were washedwith Ca²⁺ and Mg²⁺ free PBS (Gibco), subcultured further by plating thecells at a density of 2000-3000 cells/cm2 in the same media and removinghalf of the media and replacing it with fresh media twice a week untilnear confluence.

Experimental Procedures

Flow cytometric analysis of mesenchymal stem cell phenotype. Both UBCand BM derived mesenchymal stem cells were phenotyped by flow cytometry(FACSCalibur, Becton Dickinson). Fluorescein isothicyanate (FITC) orphycoerythrin (PE) conjugated antibodies against CD13, CD14, CD29, CD34,CD44, CD45, CD49e, CD73 and HLA-ABC (all from BD Biosciences, San Jose,Calif., http://www.bdbiosciences.com), CD105 (Abeam Ltd., Cambridge, UK,http://www.abcam.com) and CD133 (Miltenyi Biotec) were used for directlabeling. Appropriate FITC- and PE-conjugated isotypic controls (BDBiosciences) were used. Unconjugated antibodies against CD90 and HLA-DR(both from BD Biosciences) were used for indirect labeling. For indirectlabeling FITC-conjugated goat anti-mouse IgG antibody (Sigma-aldrich)was used as a secondary antibody.

The UBC derived cells were negative for the hematopoietic markers CD34,CD45, CD14 and CD133. The cells stained positively for the CD13(aminopeptidase N), CD29 (β1-integrin), CD44 (hyaluronate receptor),CD73 (SH3), CD90 (Thyl), CD105 (SH2/endoglin) and CD 49e. The cellsstained also positively for HLA-ABC but were negative for HLA-DR.BM-derived cells showed to have similar phenotype. They were negativefor CD 14, CD34, CD45 and HLA-DR and positive for CD13, CD29, CD44,CD90, CD105 and HLA-ABC.

Adipogenic differentiation. To assess the adipogenic potential of theUCB-derived MSCs the cells were seeded at the density of 3×10³/cm² in24-well plates (Nunc) in three replicate wells. UCB-derived MSCs werecultured for five weeks in adipogenic inducing medium which consisted ofDMEM low glucose, 2% FCS (both from Gibco), 10 μg/ml insulin, 0.1 mMindomethacin, 0.1 μM dexamethasone (Sigma-Aldrich) andpenicillin-streptomycin (Gibco) before samples were prepared for glycomeanalysis. The medium was changed twice a week during differentiationculture.

Osteogenic differentiation. To induce the osteogenic differentiation ofthe BM-derived MSCs the cells were seeded in their normal proliferationmedium at a density of 3×10³/cm² on 24-well plates (Nunc). The next daythe medium was changed to osteogenic induction medium which consisted ofα-MEM (Gibco) supplemented with 10% FBS (Gibco), 0.1 μM dexamethasone,10 mM β-glycerophosphate, 0.05 mM L-ascorbic acid-2-phosphate(Sigma-Aldrich) and penicillin-streptomycin (Gibco). BM-derived MSCswere cultured for three weeks changing the medium twice a week beforepreparing samples for glycome analysis.

Cell harvesting for glycome analysis. 1 ml of cell culture medium wassaved for glycome analysis and the rest of the medium removed byaspiration. Cell culture plates were washed with PBS buffer pH 7.2. PBSwas aspirated and cells scraped and collected with 5 ml of PBS (repeatedtwo times). At this point small cell fraction (10 μl) was taken forcell-counting and the rest of the sample centrifuged for 5 minutes at400 g. The supernatant was aspirated and the pellet washed in PBS for anadditional 2 times.

The cells were collected with 1.5 ml of PBS, transferred from 50 ml tubeinto 1.5 ml collection tube and centrifuged for 7 minutes at 5400 rpm.The supernatant was aspirated and washing repeated one more time. Cellpellet was stored at −70° C. and used for glycome analysis.

Lectin stainings. FITC-labeled Maackia amurensis agglutinin (MAA) waspurchased from EY Laboratories (USA) and FITC-labeled Sambucus nigraagglutinin (SNA) was purchased from Vector Laboratories (UK). Bonemarrow derived mesenchymal stem cell lines were cultured as describedabove. After culturing, cells were rinsed 5 times with PBS (10 mM sodiumphosphate, pH 7.2, 140 mM NaCl) and fixed with 4% PBS-bufferedparaformaldehyde pH 7.2 at room temperature (RT) for 10 minutes. Afterfixation, cells were washed 3 times with PBS and non-specific bindingsites were blocked with 3% HSA-PBS (FRC Blood Service, Finland) or 3%BSA-PBS (>99% pure BSA, Sigma) for 30 minutes at RT. According tomanufacturers' instructions cells were washed twice with PBS, TBS (20 mMTris-HCl pH 7.5, 150 mM NaCl, 10 mM CaCl₂) or HEPES-buffer (10 mM HEPES,pH 7.5, 150 mM NaCl) before lectin incubation. FITC-labeled lectins werediluted in 1% HSA or 1% BSA in buffer and incubated with the cells for60 minutes at RT in the dark. Furthermore, cells were washed 3 times 10minutes with PBS/TBS/HEPES and mounted in Vectashield mounting mediumcontaining DAPI-stain (Vector Laboratories, UK). Lectin stainings wereobserved with Zeiss Axioskop 2 plus—fluorescence microscope (Carl ZeissVision GmbH, Germany) with FITC and DAPI filters. Images were taken withZeiss AxioCam MRc-camera and with AxioVision Software 3.1/4.0 (CarlZeiss) with the 400× magnification.

Results

Glycan isolation from mesenchymal stem cell populations. The presentresults are produced from two cord blood derived mesenchymal stem celllines and cells induced to differentiate into adipogenic direction, andtwo marrow derived mesenchymal stem cell lines and cells induced todifferentiate into osteogenic direction. The caharacterization of thecell lines and differentiated cells derived from them are describedabove. N-glycans were isolated from the samples, and glycan profileswere generated from MALDI-TOF mass spectrometry data of isolated neutraland sialylated N-glycan fractions as described in the precedingexamples.

Cord Blood Derived Mesenchymal Stem Cell (CB MSC) Lines

Neutral N-glycan profiles. Neutral N-glycan profiles obtained from twoCB MSC lines are presented in FIG. 9. The two cell lines resembleclosely each other with respect to their overall neutral N-glycanprofiles. However, minor differences between the profiles are observed,and some glycan signals can only be observed in one cell line,indicating that the two cell lines have glycan structures that differthem from each other. The analysis revealed in each cell type therelative proportions of about 50-70 glycan signals that were assigned asnon-sialylated N-glycan components. Typically, significant differencesin the glycan profiles between cell populations are consistentthroughout multiple experiments.

Neutral N-glycan structural features. Neutral N-glycan groupingsproposed for the two CB MSC lines resemble each other closely,indicating that there are no major differences in their neutral N-glycanstructural features. However, CB MSCs differ from the CB mononuclearcell populations, and they have for example relatively high amounts ofneutral complex-type N-glycans, as well as hybrid-type or monoantennaryneutral N-glycans, compared to other structural groups in the profiles.

Identification of soluble glycan components. Similarly to CB mononuclearcell populations, in the present analysis neutral glycan components wereidentified in all the cell types that were assigned as soluble glycansbased on their proposed monosaccharide compositions including componentsfrom the glycan group Hex₂₋₁₂HexNAc₁ (see Figures). The abundancies ofthese glycan components in relation to each other and in relation to theother glycan signals vary between individual samples and cell types.

Sialylated N-glycan profiles. Sialylated N-glycan profiles obtained fromtwo CB MSC lines are presented in FIG. 10. The two cell lines resembleclosely each other with respect to their overall sialylated N-glycanprofiles. However, minor differences between the profiles are observed,and some glycan signals can only be observed in one cell line,indicating that the two cell lines have glycan structures that differthem from each other. The analysis revealed in each cell type therelative proportions of about 50-70 glycan signals that were assigned asacidic N-glycan components. Typically, significant differences in theglycan profiles between cell populations are consistent throughoutmultiple experiments.

Differentiation-associated changes in glycan profiles. FIG. 11 shows howneutral N-glycan profiles of CB MSCs change upon differentation inadipogenic cell culture medium. The present results indicate thatrelative abundancies of several individual glycan signals as well asglycan signal groups change due to cell culture in differentiationmedium. The major change in glycan structural groups associated withdifferentation is increase in amounts of neutral complex-type N-glycans,such as signals at m/z 1663 and m/z 1809, corresponding to theHex₅HexNAc₄ and Hex₅HexNAc4dHex₁ monosaccharide compositions,respectively. Changes were also observed in sialylated glycan profiles.

Glycosidase analyses of neutral N-glycans. Specific exoglycosidasedigestions were performed on isolated neutral N-glycan fractions from CBMSC lines as described in the preceding Examples. The results ofα-mannosidase analysis are described in FIG. 12, showing in detail whichof the neutral N-glycan signals in the neutral N-glycan profiles of CBMNC lines are susceptible to α-mannosidase digestion, indicating for thepresence of non-reducing terminal α-mannose residues in thecorresponding glycan structures. As an example, the major neutralN-glycan signals at m/z 1257, 1419, 1581, 1743, and 1905, which wereprelminarily assigned as high-mannose type N-glycans according to theirproposed monosaccharide compositions Hex₅₋₉HexNAc₂, were shown tocontain terminal α-mannose residues thus confirming the preliminaryassignment. The results of β1,4-galactosidase analysis are described inFIG. 13 (for a CB MNC line) and FIG. 14 (for a CB MNC line cultured inadipogenic medium) showing in detail which of the neutral N-glycansignals in the neutral N-glycan profiles of CB MNC lines anddifferentiated CB MNCs are susceptible to β1,4-galactosidase digestion,indicating for the presence of non-reducing terminal β1,4-galactoseresidues in the corresponding glycan structures. As an example, themajor neutral complex-type N-glycan signals at m/z 1663 and m/z 1809were shown to contain terminal β1,4-linked galactose residues.

Bone Marrow Derived Mesenchymal Stem Cell (BM MSC) Lines

Neutral N-glycan profiles and differentiation-associated changes inglycan profiles. Neutral N-glycan profiles obtained from a BM MSC line,grown in proliferation medium and in osteogenic medium are presented inFIG. 15. The BM MSCs resemble CB MSC lines with respect to their overallneutral N-glycan profiles. However, differences between cell linesderived from the two sources are observed, and some glycan signals canonly be observed in one cell line, indicating that the cell lines haveglycan structures that differ them from each other. The majorcharacteristic structural feature of BM MSCs is even more abundantneutral complex-type N-glycans compared to CB MSC lines. Similarly to CBMSCs, these glycans were also the major increased glycan signal groupupon differentiation of BM MSCs. The analysis revealed in each cell typethe relative proportions of about 50-70 glycan signals that wereassigned as non-sialylated N-glycan components. Typically, significantdifferences in the glycan profiles between cell populations areconsistent throughout multiple experiments.

Sialylated N-glycan profiles. Sialylated N-glycan profiles obtained froma BM MSC line, grown in proliferation medium and in osteogenic mediumare presented in FIG. 16. The undifferentiated and differentiated cellsresemble closely each other with respect to their overall sialylatedN-glycan profiles. However, minor differences between the profiles areobserved, and some glycan signals can only be observed in one cell line,indicating that the two cell types have glycan structures that differthem from each other. The analysis revealed in each cell type therelative proportions of about 50 glycan signals that were assigned asacidic N-glycan components. Typically, significant differences in theglycan profiles between cell populations are consistent throughoutmultiple experiments.

Sialidase analysis. The sialylated N-glycan fraction isolated from BMMSCs was digested with broad-range sialidase as described in thepreceding Examples. After the reaction, it was observed by MALDI-TOFmass spectrometry that the vast majority of the sialylated N-glycanswere desialylated and transformed into corresponding neutral N-glycans,indicating that they had contained sialic acid residues (NeuAc and/orNeuGc) as suggested by the proposed monosaccharide compositions. FIG. 17shows the glycan profiles of combined neutral and desialylated(originally sialylated) N-glycan fractions of BM MSCs grown inproliferation medium and in osteogenic medium. The profiles correspondto total N-glycan profiles isolated from the cell samples (indesialylated form). It is calculated that in undifferentiated BM MSCs(grown in osteogenic medium), approximately 53% of the N-glycan signalscorrespond to high-mannose type N-glycan monosaccharide compositions, 8%to low-mannose type N-glycans, 31% to complex-type N-glycans, and 7% tohybrid-type or monoantennary N-glycan monosaccharide compositions. Indifferentiated BM MSCs (grown in osteogenic medium), approximately 28%of the N-glycan signals correspond to high-mannose type N-glycanmonosaccharide compositions, 9% to low-mannose type N-glycans, 50% tocomplex-type N-glycans, and 11% to hybrid-type or monoantennary N-glycanmonosaccharide compositions.

Lectin binding analysis of mesenchymal stem cells. As described underExperimental procedures, bone marrow derived mesenchymal stem cells wereanalyzed for the presence of ligands of α2,3-linked sialic acid specific(MAA) and α2,6-linked sialic acid specific (SNA) lectins on theirsurface. It was revealed that MAA bound strongly to the cells whereasSNA bound weakly, indicating that in the cell culture conditions, thecells had significantly more α2,3-linked than α2,6-linked sialic acidson their surface glycoconjugates. The present results suggest thatlectin staining can be used as a further means to distinguish differentcell types and complements mass spectrometric profiling results.

Detection of Potential Glycan Contaminations from Cell Culture Reagents

In the sialylated N-glycan profiles of MSC lines, specific N-glycansignals were observed that indicated contamination of mesenchymal stemcell glycoconjugates by abnormal sialic acid residues. First, when thecells were cultured in cell culture media with added animal sera, suchas bovine of equine sera, potential contamination byN-glycolylneuraminic acid (NeuSGc) was detected. The glycan signals atm/z 1946, corresponding to the [M-H]⁻ ion of NeuGc₁Hex₅HexNAc₄, as wellas m/z 2237 and m/z 2253, corresponding to the [M-H]⁻ ions ofNeuGc₁NeuAc₁Hex₅HexNAC₄ and NeuGC₂Hex₅HexNAc₄, respectively, wereindicative of the presence of Neu5Gc, i.e. a sialic acid residue with 16Da larger mass than N-acetylneuraminic acid (Neu5Ac). Moreover, when thecells were cultured in cell culture media with added horse serum,potential contamination by O-acetylated sialic acids was detected.Diagnostic signals used for detection of O-acetylated sialic acidcontaining sialylated N-glycans included [M-H]⁻ ions ofAc₁NeuAc₂Hex₅HexNAc₄, Ac₂NeuAC₂Hex₅HexNAc₄, and Ac₂NeuAc₂Hex₅HexNAc₄, atcalculated m/z 1972.7, 2263.8, and 2305.8, respectively.

Conclusions

Uses of the glycan profiling method. The results indicate that thepresent glycan profiling method can be used to differentiate CB MSClines and BM MSC lines from each other, as well as from other cell typessuch as cord blood mononuclear cell populations. Differentation-inducedchanges as well as potential glycan contaminations from e.g. cellculture media can also be detected in the glycan profiles, indicatingthat changes in cell status can be detected by the present method. Themethod can also be used to detect MSC-specific glycosylation featuresincluding those discussed below.

Differences in glycosylation between cultured cells and native humancells. The present results indicate that BM MSC lines have morehigh-mannose type N-glycans and less low-mannose type N-glycans comparedto the other N-glycan structural groups than mononuclear cells isolatedfrom cord blood. Taken together with the results obtained from culturedhuman embryonal stem cells in the following Examples, it is indicatedthat this is a general tendency of cultured stem cells compared tonative isolated stem cells. However, differentiation of BM MSCs inosteogenic medium results in significantly increased amounts ofcomplex-type N-glycans and reduction in the amounts of high-mannose typeN-glycans.

Mesenchymal stem cell line specific glycosylation features. The presentresults indicate that mesenchymal stem cell lines differ from the othercell types studied in the present study with regard to specific featuresof their glycosylation, such as:

-   1) Both CB MSC lines and BM MSC lines have unique neutral and    sialylated N-glycan profiles;-   2) The major characteristic structural feature of both CB and BM MSC    lines is abundant neutral complex-type N-glycans;-   3) An additional characteristic feature is low sialylation level of    complex-type N-glycans.

Example 9 MALDI-TOF Mass Spectrometric N-glycan Profiling of HumanEmbryonic Stem Cell Lines Examples of Cell Material Production

Human Embryonic Stem Cell Lines (hESC)

Undifferentiated hESC. Processes for generation of hESC lines fromblastocyst stage in vitro fertilized excess human embryos have beendescribed previously (e.g. Thomson et al., 1998). Two of the analysedcell lines in the present work were initially derived and cultured onmouse embryonic fibroblasts feeders (MEF; 12-13 pc fetuses of the ICRstrain), and two on human foreskin fibroblast feeder cells (HFF;CRL-2429 ATCC, Mananas, USA). For the present studies all the lines weretransferred on HFF feeder cells treated with mitomycin-C (1 μg/ml;Sigma-Aldrich) and cultured in serum-free medium (Knockout™ D-MEM;Gibco® Cell culture systems, Invitrogen, Paisley, UK) supplemented with2 mM L-Glutamin/Penicillin streptomycin (Sigma-Aldrich), 20% KnockoutSerum Replacement (Gibco), 1× non-essential amino acids (Gibco), 0.1 mMβ-mercaptoethanol (Gibco), 1×ITSF (Sigma-Aldrich) and 4 ng/ml bFGF(Sigma/Invitrogen).

Stage 2 differentiated hESC (embryoid bodies). To induce the formationof embryoid bodies (EB) the hESC colonies were first allowed to grow for10-14 days whereafer the colonies were cut in small pieces andtransferred on non-adherent Petri dishes to form suspension cultures.The formed EBs were cultured in suspension for the next 10 days instandard culture medium (see above) without bFGF.

Stage 3 differentiated hESC. For further differentiation EBs weretransferred onto gelatin-coated (Sigma-Aldrich) adherent culture dishesin media consisting of DMEM/F12 mixture (Gibco) supplemented with ITS,Fibronectin (Sigma), L-glutamine and antibiotics. The attached cellswere cultured for 10 days whereafter they were harvested.

Sample preparation. The cells were collected mechanically, washed, andstored frozen prior to glycan analysis.

Results

Neutral N-glycan profiles—effect of differentiation status. NeutralN-glycan profiles obtained from a human embryonal stem cell (hESC) line,its embryoid body (EB) differentiated form, and its stage 3 (st.3)differentiated form are presented in FIG. 18. Although the cell typesresemble each other with respect to the major neutral N-glycan signals,the neutral N-glycan profiles of the two differentiated cell formsdiffer significantly from the undifferentiated hESC profile. In fact,the farther differentiated the cell type is, the more its neutralN-glycan profile differs from the undifferentiated hESC profile.Multiple differences between the profiles are observed, and many glycansignals can only be observed in one or two out of three cell types,indicating that differentiation induces the appearance of new glycantypes. The analysis revealed in each cell type the relative proportionsof about 40-55 glycan signals that were assigned as non-sialylatedN-glycan components. Typically, significant differences in the glycanprofiles between cell populations are consistent throughout multipleexperiments.

Neutral N-glycan profiles—comparison of hESC lines. Neutral N-glycanprofiles obtained from four hESC lines are presented in FIG. 20. Thefour cell lines closely resemble each other. Individual profilecharacteristics and cell line specific glycan signals are present in theglycan profiles, but it is concluded that hESC lines resemble more eachother with respect to their neutral N-glycan profiles and are differentfrom differentiated EB and st3 cell types. hESC lines 3 and 4 arederived from sibling embryos, and their neutral N-glycan profilesresemble more each other and are different from the two other celllines, i.e. they contain common glycan signals. The analysis revealed ineach cell type the relative proportions of about 40-55 glycan signalsthat were assigned as non-sialylated N-glycan components. Typically,significant differences in the glycan profiles between cell populationsare consistent throughout multiple experiments.

Neutral N-glycan structural features. Neutral N-glycan groupingsproposed for analysed cell types are presented in Table 12. Again, theanalysed three major cell types, namely undifferentiated hESCs,differentiated cells, and human fibroblast feeder cells, differ fromeach other significantly. Within each cell type, however, there areminor differences between individual cell lines. Moreover,differentiation-associated neutral N-glycan structural features areexpressed more strongly in st.3 differentiated cells than in EB cells.Cell-type specific glycosylation features are discussed below inConclusions.

Glycosidase analysis of neutral N-glycan fractions. Specificexoglycosidase digestions were performed on isolated neutral N-glycanfractions from hESC lines as described in the preceding Examples. Inα-mannosidase analysis, several neutral glycan signals were shown to besusceptible to α-mannosidase digestion, indicating for potentialpresence of non-reducing terminal α-mannose residues in thecorresponding glycan structures. In hESC and EB cells, these signalsincluded m/z 917, 1079, 1095, 1241, 1257, 1378, 1393, 1403, 1444, 1555,1540, 1565, 1581, 1606, 1622, 1688, 1743, 1768, 1905, 1996, 2041, 2067,2158, and 2320 (the corresponding monosaccharide compositions arepresented in for example Table 1). In β1,4-galactosidase analysis,several neutral glycan signals were shown to be susceptible toβ1,4-galactosidase digestion, indicating for potential presence ofnon-reducing terminal β1,4-galactose residues in the correspondingglycan structures. In hESC and EB cells, these signals included m/z 609,771, 892, 917, 1241, 1378, 1393, 1555, 1565, 1606, 1622, 1647, 1663,1704, 1809, 1850, 1866, 1955, 1971, 1996, 2012, 2028, 2041, 2142, 2174,and 2320 (the corresponding monosaccharide compositions are presented infor example Table 1). In α1,3/4-fucosidase analysis, several neutralglycan signals were shown to be susceptible to α1,3/4-fucosidasedigestion, indicating for potential presence of non-reducing terminalα1,3- and/or α1,4-fucose residues in the corresponding glycanstructures. In hESC and EB cells, these signals included m/Z 1120, 1590,1784, 1793, 1955, 1996, 2101, 2117, 2142, 2158, 2190, 2215, 2247, 2263,2304, 2320, 2393, and 2466 (the corresponding monosaccharidecompositions are presented in for example Table 1).

Identification of soluble glycan components. Similarly to the cell typesdescribed in the preceding examples, in the present analysis neutralglycan components were identified in all the cell types that wereassigned as soluble glycans based on their proposed monosaccharidecompositions including components from the glycan group Hex₂₋₁₂HexNAc₁(see Figures). The abundancies of these glycan components in relation toeach other and in relation to the other glycan signals vary betweenindividual samples and cell types.

Sialylated N-glycan profiles—effect of differentiation status.Sialylated N-glycan profiles obtained from a human embryonal stem cell(hESC) line, its embryoid body (EB) differentiated form, and its stage 3(st.3) differentiated form are presented in FIG. 19. Although the celltypes resemble each other with respect to the major sialylated N-glycansignals, the sialylated N-glycan profiles of the two differentiated cellforms differ significantly from the undifferentiated hESC profile. Infact, the farther differentiated the cell type is, the more itssialylated N-glycan profile differs from the undifferentiated hESCprofile. Multiple differences between the profiles are observed, andmany glycan signals can only be observed in one or two out of three celltypes, indicating that differentiation induces the appearance of newglycan types as well as decrease in amounts of stem cell specific glycantypes. For example, there is significant differentation-associateddecrease in relative amounts of glycan signals at m/z 1946 and 2222,corresponding to monosaccharide compositions NeuGc₁Hex₅HexNAc₄ andNeuAc₁Hex₅HexNAc₄dHex₂, respectively. The analysis revealed in each celltype the relative proportions of about 50-70 glycan signals that wereassigned as acidic N-glycan components. Typically, significantdifferences in the glycan profiles between cell populations areconsistent throughout multiple experiments.

Sialylated N-glycan profiles—comparison of hESC lines. SialylatedN-glycan profiles obtained from four hESC lines are presented in FIG.21. The four cell lines closely resemble each other. Individual profilecharacteristics and cell line specific glycan signals are present in theglycan profiles, but it is concluded that hESC lines resemble more eachother with respect to their sialylated N-glycan profiles and aredifferent from differentiated EB and st.3 cell types. The analysisrevealed in each cell type the relative proportions of about 50-70glycan signals that were assigned as acidic N-glycan components.Typically, significant differences in the glycan profiles between cellpopulations are consistent throughout multiple experiments.

Human fibroblast feeder cell lines. Sialylated N-glycan profilesobtained from human fibroblast feeder cell lines are presented in FIG.22. The present results show that the feeder cells differ from hESC, EB,and st3 differentiated cells, and that feeder cells grown separately andwith hESC cells differ from each other.

Sialylated N-glycan structuralfeatures. Sialylated N-glycan groupingsproposed for analysed cell types are presented in Table 13. Again, theanalysed three major cell types, namely undifferentiated hESCs,differentiated cells, and human fibroblast feeder cells, differ fromeach other significantly. Within each cell type, however, there areminor differences between individual cell lines. Moreover,differentiation-associated sialylated N-glycan structural features areexpressed more strongly in st.3 differentiated cells than in EB cells.Cell-type specific glycosylation features are discussed below inConclusions.

Conclusions

Comparison of glycan profiles. Differences in the glycan profilesbetween cell types were consistent throughout multiple samples andexperiments, indicating that the present method of glycan profiling andthe differences in the present glycan profiles can be used to identifyhESCs or cells differentiated therefrom, or other cells such as feedercells, or to determine their purity, or to identify cell types presentin a sample. The present method and the present results can also be usedto identify cell-type specific glycan structural features or cell-typespecific glycan profiles. The method proved especially useful indetermination of differentiation stage, as demonstrated by comparinganalysis results between hESC, EB, and st.3 differentiated cells.Furthermore, hESCs were shown to have unique glycosylation profiles,which can be differentiated from differentiated cell types as well asfrom other stem cell types such as MSCs, indicating that stem cells ingeneral and also specific stem cell types can be identified by thepresent method. The present method could also detect glycan structurescommon to hESC lines derived from sibling embryos, indicating thatrelated structural features can be identified in different cell lines ortheir similarity be estimated by the present method.

Comparison of neutral N-glycan structural features. Differences inglycosylation profiles between analyzed cell types were identified basedon proposed structural features, which can be used to identify cell-typespecific glycan structural features. Identified cell-type specificfeatures of neutral N-glycan profiles are concluded below:

hESC Lines:

-   1) Increased amounts of fucosylated neutral N-glycans, especially    glycans with two or more deoxyhexose residues per chain, indicating    increased expression of neutral N-glycans containing α1,2-, α1,3-,    or α1,4-linked fucose residues; and-   2) Increased amounts of larger neutral N-glycans.

EBs and st3 Differentiated Cells (st.3 Cells Expressing the FeaturesMore Strongly):

-   1) Lower amounts of neutral N-glycans containing two or more    deoxyhexose residues per chain, indicating reduced expression of    neutral N-glycans containing α1,2-, α1,3-, or α1,4-linked fucose    residues;-   2) Increased amounts of hybrid-type, monoantennary, and complex-type    neutral N-glycans.-   3) Increased amounts of terminal HexNAc residues; and-   4) Potentially increased amounts of bisecting GlcNAc structures.

Human Fibroblast Feeder Cells:

-   1) Increased amounts of larger neutral N-glycans;-   2) Lower amounts of neutral N-glycans containing two or more    deoxyhexose residues per chain, indicating reduced expression of    neutral N-glycans containing α1,2-, α1,3-, or α1,4-linked fucose    residues;-   3) Increased amounts of terminal HexNAc residues; and-   4) Potentially no bisecting GlcNAc structures.

Comparison of sialylated N-glycan structural features. Differences inglycosylation profiles between analyzed cell types were identified basedon proposed structural features, which can be used to identify cell-typespecific glycan structural features. Identified cell-type specificfeatures of sialylated N-glycan profiles are concluded below:

hESC Lines:

-   1) Increased amounts of fucosylated sialylated N-glycans, especially    glycans with two or more deoxyhexose residues per chain, indicating    increased expression of sialylated N-glycans containing α1,2-,    α1,3-, or α1,4-linked fucose residues;-   2) Increased amounts of terminal HexNAc residues; and-   3) Increased amounts of Neu5Gc containing sialylated N-glycans.

EBs and st3 Differentiated Cells (st3 Cells Expressing the Features MoreStrongly):

-   1) Lower amounts of sialylated N-glycans containing two or more    deoxyhexose residues per chain, indicating reduced expression of    sialylated N-glycans containing α1,2-, α1,3-, or α1,4-linked fucose    residues;-   2) Increased amounts of hybrid-type or monoantennary sialylated    N-glycans; and-   3) Potentially increased amounts of bisecting GlcNAc structures.

Human Fibroblast Feeder Cells:

-   1) Increased amounts of larger sialylated N-glycans;-   2) Lower amounts of terminal HexNAc residues; and-   3) Potentially lower amounts of bisecting GlcNAc structures.

Example 10 Enzymatic Modification of Cell Surface Glycan StructuresExperimental Procedures

Enzymatic modifications. Sialyltransferase reaction: Human cord bloodmononuclear cells (3×10⁶ cells) were modified with 60 mUα2,3-(N)-sialyltransferase (rat, recombinant in S. frugiperda,Calbiochem), 1.6 μmol CMP-Neu5Ac in 50 mM sodium3-morpholinopropanesulfonic acid (MOPS) buffer pH 7.4, 150 mM NaCl attotal volume of 100 μl for up to 12 hours. Fucosyltransferase reaction:Human cord blood mononuclear cells (3×10⁶ cells) were modified with 4 mUα1,3-fucosyltransferase VI (human, recombinant in S. frugiperda,Calbiochem), 1 μmol GDP-Fuc in 50 mM MOPS buffer pH 7.2, 150 mM NaCl attotal volume of 100 μl for up to 3 hours. Broad-range sialidasereaction: Human cord blood mononuclear cells (3×10⁶ cells) were modifiedwith 5 mU sialidase (A. ureafaciens, Glyko, UK) in 50 mM sodium acetatebuffer pH 5.5, 150 mM NaCl at total volume of 100 μl for up to 12 hours.α2,3-specific sialidase reaction: Cells were modified withα2,3-sialidase (S. pneumoniae, recombinant in E. coli) in 50 mM sodiumacetate buffer pH 5.5, 150 mM NaCl at total volume of 100 μl.α-mannosidase reaction: α-mannosidase was from Jack beans and reactionwas performed essentially similarly as with other enzymes describedabove. Sequential enzymatic modifications: Between sequential reactionscells were pelleted with centrifugation and supernatant was discarded,after which the next modification enzyme in appropriate buffer andsubstrate solution was applied to the cells as described above. Washingprocedure: After modification, cells were washed with phosphate bufferedsaline.

Glycan analysis. After washing the cells, total cellular glycoproteinswere subjected to N-glycosidase digestion, and sialylated and neutralN-glycans isolated and analyzed with mass spectrometry as describedabove. For O-glycan analysis, the glycoproteins were subjected toreducing alkaline β-elimination essentially as described previously(Nyman et al., 1998), after which sialylated and neutral glycan alditolfractions were isolated and analyzed with mass spectrometry as describedabove.

Results

Sialidase digestion. Upon broad-range sialidase catalyzed desialylationof living cord blood mononuclear cells, sialylated N-glycan structuresas well as O-glycan structures (data not shown) were desialylated, asindicated by increase in relative amounts of corresponding neutralN-glycan structures, for example Hex₆HexNAc₃, Hex₅HexNAc₄dHex₀₋₂, andHex₆HexNAc₅dHex₀₋₁ monosaccharide compositions (Table 15). In general, ashift in glycosylation profiles towards glycan structures with lesssialic acid residues was observed in sialylated N-glycan analyses uponbroad-range sialidase treatment. The shift in glycan profiles of thecells upon the reaction served as an effective means to characterize thereaction results. It is concluded that the resulting modified cellscontained less sialic acid residues and more terminal galactose residuesat their surface after the reaction.

α2,3-specific sialidase digestion. Similarly, upon α2,3-specificsialidase catalyzed desialylation of living mononuclear cells,sialylated N-glycan structures were desialylated, as indicated byincrease in relative amounts of corresponding neutral N-glycanstructures (data not shown). In general, a shift in glycosylationprofiles towards glycan structures with less sialic acid residues wasobserved in sialylated N-glycan analyses upon α2,3-specific sialidasetreatment. The shift in glycan profiles of the cells upon the reactionserved as an effective means to characterize the reaction results. It isconcluded that the resulting modified cells contained less α2,3-linkedsialic acid residues and more terminal galactose residues at theirsurface after the reaction.

Sialyltransferase reaction. Upon α2,3-sialyltransferase catalyzedsialylation of living cord blood mononuclear cells, numerous neutral(Table 15) and sialylated N-glycan (Table 14) structures as well asO-glycan structures (data not shown) were sialylated, as indicated bydecrease in relative amounts of neutral N-glycan structures(Hex₅HexNAc₄dHex₀₋₃ and Hex₆HexNAc₅dHex₀₋₂ monosaccharide compositionsin Table 15) and increase in the corresponding sialylated structures(for example the NeuAc₂Hex₅HexNAc₄dHex, glycan in Table 14). In general,a shift in glycosylation profiles towards glycan structures with moresialic acid residues was observed both in N-glycan and O-glycananalyses. It is concluded that the resulting modified cells containedmore α2,3-linked sialic acid residues and less terminal galactoseresidues at their surface after the reaction.

Fucosyltransferase reaction. Upon α1,3-fucosyltransferase catalyzedfucosylation of living cord blood mononuclear cells, numerous neutral(Table 15) and sialylated N-glycan structures as well as O-glycanstructures (see below) were fucosylated, as indicated by decrease inrelative amounts of nonfucosylated glycan structures (without dhex inthe proposed monosaccharide compositions) and increase in thecorresponding fucosylated structures (with n_(dHex)>0 in the proposedmonosaccharide compositions). For example, before fucosylation O-glycanalditol signals at m/z 773, corresponding to the [M+Na]⁺ ion ofHex₂HexNAc2 alditol, and at m/z 919, corresponding to the [M+Na]⁺ ion ofHex₂HexNAc₂dHex₁ alditol, were observed in approximate relativeproportions 9:1, respectively (data not shown). After fucosylation, theapproximate relative proportions of the signals were 3:1, indicatingthat significant fucosylation of neutral O-glycans had occurred. Somefucosylated N-glycan structures were even observed after the reactionthat had not been observed in the original cells, for example neutralN-glycans with proposed structures Hex₆HexNAc₅dHex₁ and Hex₆HexNAc₅dHex₂(Table 15), indicating that in α1,3-fucosyltransferase reaction the cellsurface of living cells can be modified with increased amounts orextraordinary structure types of fucosylated glycans, especiallyterminal Lewis x epitopes in protein-linked N-glycans as well as inO-glycans.

Sialidase digestion followed by sialyltransferase reaction. Cord bloodmononuclear cells were subjected to broad-range sialidase reaction,after which α2,3-sialyltransferase and CMP-Neu5Ac were added to the samereaction, as described under Experimental procedures. The effects ofthis reaction sequence on the N-glycan profiles of the cells aredescribed in FIG. 23. The sialylated N-glycan profile was also analyzedbetween the reaction steps, and the result clearly indicated that sialicacids were first removed from the sialylated N-glycans (indicated forexample by appearance of increased amounts of neutral N-glycans), andthen replaced by α2,3-linked sialic acid residues (indicated for exampleby disappearance of the newly formed neutral N-glycans; data not shown).It is concluded that the resulting modified cells contained moreα2,3-linked sialic acid residues after the reaction.

Sialyltransferase reaction followed by fucosyltransferase reaction. Cordblood mononuclear cells were subjected to α2,3-sialyltransferasereaction, after which α1,3-fucosyltransferase and GDP-fucose were addedto the same reaction, as described under Experimental procedures. Theeffects of this reaction sequence on the sialylated N-glycan profiles ofthe cells are described in FIG. 24. The results show that a major partof the glycan signals (detailed in Table 16) have undergone changes intheir relative intensities, indicating that a major part of thesialylated N-glycans present in the cells were substrates of theenzymes. It was also clear that the combination of the enzymaticreaction steps resulted in different result than either one of thereaction steps alone.

Different from the α1,3-fucosyltransferase reaction described above,sialylation before fucosylation apparently sialylated the neutralfucosyltransferase acceptor glycan structures present on cord bloodmononuclear cell surfaces, resulting in no detectable formation of theneutral fucosylated N-glycan structures that had emerged afterα1,3-fucosyltransferase reaction alone (discussed above; Table 15).

α-mannosidase reaction. α-mannosidase reaction of whole cells showed aminor reduction of glycan signals including those indicated to containα-mannose residues in the preceding examples.

Glycosyltranserase-derived glycan structures. We detected thatglycosylated glycosyltransferase enzymes can contaminate cells inmodification reactions. For example, when cells were incubated withrecombinant fucosyltransferase or sialyltransferase enzymes produced inS. frugiperda cells, N-glycosidase and mass spectrometric analysis ofcellular and/or cell-associated glycoproteins resulted in detection ofan abundant neutral N-glycan signal at m/z 1079, corresponding to[M+Na]⁺ ion of Hex₃HexNAc₂dHex₁ glycan component (calc. m/z 1079.38).Typically, in recombinant glycosyltransferase treated cells, this glycansignal was more abundant than or at least comparable to the cells' ownglycan signals, indicating that insect-derived glycoconjugates are avery potent contaminant associated with recombinant glycan-modifiedenzymes produced in insect cells. Moreover, this glycan contaminationpersisted even after washing of the cells, indicating that theinsect-type glycoconjugate corresponding to or associated with theglycosyltransferase enzymes has affinity towards cells or has tendencyto resist washing from cells. To confirm the origin of the glycansignal, we analyzed glycan contents of commercial recombinantfucosyltransferase and sialyltransferase enzyme preparations and foundthat the m/z 1079 glycan signal was a major N-glycan signal associatedwith these enzymes. Corresponding N-glycan structures, e.g.Manα3(Manα6)Manβ4GlnNAc(Fucα3/6)GlcNAc(β-N-Asn), have been describedpreviously from glycoproteins produced in S. frugiperda cells(Staudacher et al., 1992; Kretzchmar et al., 1994; Kubelka et al., 1994;Altmann et al., 1999). As described in the literature, these glycanstructures, as well as other glycan structures potentially contaminatingcells treated with recombinant or purified enzymes, especiallyinsect-derived products, are potentially immunogenic in humans and/orotherwise harmful to the use of the modified cells. It is concluded thatglycan-modifying enzymes must be carefully selected for modification ofhuman cells, especially for clinical use, not to contain immunogenicglycan epitopes, non-human glycan structures, and/or other glycanstructures potentially having unwanted biological effects.

Example 11 MALDI-TOF Mass Spectrometric Profiling of Cell SurfaceGlycans Experimental Procedures and Results

Cells, Mononuclear cells were isolated from human peripheral blood byFicoll-Hypaque density gradient (Amersham Biosciences, Piscataway, USA)essentially as described. The surface glycoprotein glycans wereliberated by mild trypsin treatment (80 micrograms/ml in PBS) at +37degrees Celsius for 2 hours. The intact cells were harvested bycentrifugation, and the supernatant containing the liberated glycans (atthis stage as cell surface glycoprotein glycopeptides) was taken forfurther analyses. The harvested cells and the supernatant were subjectedto Glycan profiling by protein N-glycosidase as described in thepreceding examples. The N-glycan profiles of the supernatant containingthe cell surface glycoprotein glycopeptides, were compared againstN-glycan profiles of the cells harvested from the trypsin treatment.

Results

N-Glycan analyses of HMC cell surface glycopeptide glycomes. HMC wereisolated from peripheral blood, treated with trypsin to release thesurface glycoprotein glycopeptides, followed by release of glycopeptideglycans, and subjected to glycome profiling as described underExperimental procedures. In MALDI-TOF mass spectrometry of thesialylated N-glycan fractions, several glycon signals were detected inthese samples. When the resulting glycome profile was compared to acorresponding glycome isolated from the trypsin treated cells, it couldbe observed that many sialylated components were enriched in the surfaceglycoprotein glycopeptide fraction, whereas some structures appeared tohave more intracellular localization. Examples or the former structuresare (monosaccharide compositions in parenthesis): m/z [M-H]⁺ 1930(SaHex5HexNAc4), 2221 (Sa2Hex5HexNAc4), 2222 (SaHex5HexNAc4dHex2), 2367(Sa2Hex5HexNAc4dHex), 2368(SaHex5HexNAc4dHex3), 2587(SaHex6HexNAc5dHex2), and 3024 (Sa3Hex6HexNAc5dHex). Examples of thelatter are m/z 1873(SaHex5HexNAc3dHex), and 2035(SaHexHexNAc3dHex).

Example 12 Comparison of Human and Murine Fibroblast Feeder CellN-glycan Profiles Results

N-glycans were isolated, divided into sialylated and neutral fractions,and analysed by MALDI-TOF mass spectrometry as described in thepreceding Examples. Comparison of sialylated N-glycan profiles of humanfibroblast feeder cells and mouse fibroblast feeder cells is shown inFIG. 25. There are numerous differences in the glycan profiles and it isconcluded that human and murine feeder cells differ from each othersignificantly with respect to their overall glycan profiles as well asmany individual glycan signals. The major differences are 2092 and 2238,corresponding to the monosaccharide compositions NeuAc₁Hex₆HexNAc₄ andNeuAc₁Hex₆HexNAc₄dHex₁, respectively. These signals correspond to themajor sialylated N-glycans that human embryonal stem cells interact withon the cell surfaces of their feeder cells. The present results indicatethat the glycan analysis method can be used to study species-specificdifferences in stem cell to feeder cell interactions.

Example 13 Proton NMR Analysis of Human Embryonic Stem Cell N-glycanFractions Experimental Procedures

N-glycans were isolated from human embryonic stem cell (hESC) line (25million cells) and fractionated into neutral and acidic N-glycanfractions as described above. The final purification prior to NMRanalysis was performed by gel filtration high-performance liquidchromatography (HPLC) on a Superdex Peptide HR10/300 column in water or50 mM ammonium bicarbonate for the neutral and acidic fractions,respectively. Fractions were collected and MALDI-TOF mass spectra wererecorded from each fraction as described above (data not shown). Allfractions containing N-glycans were pooled and prepared for the NMRexperiment. The yields of neutral and acidic glycans were 4.0 and 6.6nmol, respectively.

Prior to NMR analysis the purified glycome fractions were repeatedlydissolved in 99.996% deuterium oxide and dried to omit H₂O and toexchange sample protons. The ¹H-NMR spectra at 800 MHz were recordedusing a cryo-probe for enhanced sensitivity. Chemical shifts areexpressed in parts per million (ppm) by reference to internal standardacetone (2.225 ppm).

Results And Discussion

Neutral N-glycan fraction. The identified signals in the neutralN-glycan spectrum are described in Table 17. The identified signals wereconsistent with N-glycan structures, more specifically high-mannose typeN-glycan structures such as the structures A-D in FIG. 26 that have theproposed monosaccharide compositions Man₇₋₉GlcNAc₂. In the mass spectrumrecorded from the pooled neutral N-glycan fraction, the signals with theHex₇₋₉HexNAc₂ composition together accounted for more than a half of thetotal signal intensity, which is consistent with the NMR result thatthese signals were the major glycans in the sample. The NMR spectrumcontained the characteristic signals of the glycan structures A-D (Fu etal., 1994; Hard et al., 1991) and the significant signals in the NMRspectrum can be explained by the following glycan structurecombinations: A+D, B+C, A+B+D, A+C+D, B+C+D, and A+B+C+D.

Neutral N-glycan core sequences. The identified N-glycan core structurecommon to all the identified glycan structures in the NMR spectrumincludes the following glycan sequences: the internal core sequencesManβ4GlcNAc, Manα3Manβ4GlcNAc, Manα6Manβ4GlcNAc, andManα3(Manα6)Manβ4GlcNAc, and the reducing terminal glycan core sequencesGlcNAcβ4GlcNAc, Manβ4GlcNAcβ4GlcNAc, Manα3Manβ4GlcNAcβ4GlcNAc,Manα6Manβ4GlcNAcβ4GlcNAc, and Manα3(Manα6)Manβ4GlcNAcβ4GlcNAc. TheN-glycans in the sample were liberated by N-glycosidase F enzymeindicating that the reducing terminal core sequences were β-N-linked toasparagine residues in the original sample glycoproteins. Other glycancore structures could not be identified in the spectrum.

Neutral N-glycan antennae. In the identified structures A-D, the commonreducing terminal N-glycan core sequence Manα3(Manα6)Manβ4GlcNAcβ4GlcNAcis further elongated by the following antennae: Manα2Manα2 or Manα2 tothe α3-linked Man; and/or Manα2Manα3, Manα2Manα6, Manα3, and/or Manα6 tothe α6-linked Man. Other glycan antennae could not be identified in thespectrum.

Acidic N-glycan fraction. The identified signals in the acidic N-glycanspectrum are described in Table 18. The identified signals wereconsistent with N-glycan structures, more specifically complex typeN-glycan structures such as the reference structures A-E in FIG. 27(H{dot over (a)}rd et al., 1992; Helin et al., 1995). In the massspectrum recorded from the pooled acidic N-glycan fraction, the signalscontaining exactly five hexoses and four N-acetylhexosamines in theirproposed composition i.e. containing the Hex₅HexNAc₄ structural feature(like structures B-E) together accounted for approximately 45% of thetotal signal intensity, which is consistent with the NMR result that thecorresponding glycans were the major glycans in the sample. The NMRspectrum contained the characteristic signals of the structures A-E, andthe significant signals in the NMR spectrum can be explained by thestructural components of these reference structures.

Acidic N-glycan core sequences. The identified N-glycan core structurecommon to all the identified glycan structures in the NMR spectrumincludes the following glycan sequences: the reducing terminal glycancore sequences GlcNAcβ4(±Fucα6)GlcNAc, Manβ4GlcNAcβ4(±Fucα6)GlcNAc,Manα3Manβ4GlcNAcβ4(±Fucα6)GlcNAc, Manα6Manβ4GlcNAcβ4(±Fucα6)GlcNAc, andManα3(Manα6)Manβ4GlcNAcβ4(±Fucα6)GlcNAc, wherein ±Fucα6 indicates thesite of N-glycan core fucosylation. The N-glycans in the sample wereliberated by N-glycosidase F enzyme indicating that the reducingterminal core sequences were β-N-linked to asparagine residues in theoriginal sample glycoproteins. Other glycan core structures could not beidentified in the spectrum.

Acidic N-glycan antennae. In the reference structures A-D, the reducingterminal N-glycan core sequences are further elongated by the followingantennae, which were also identified in the recorded spectrum:Neu5Acα3Galβ4GlcNAcβ2, Neu5Acα6Galβ4GlcNAcβ2, Galβ4GlcNAcβ2, and/orGalα3Galβ4GlcNAcβ2 to either α3-linked Man or α6-linked Man. Theidentified antennae in the NMR spectrum include the internal glycansequence GlcNAc β-linked or more specifically β2-linked to the N-glycancore structure. Other glycan antennae could not be identified in thespectrum, indicating that these antennae were the most abundant antennastructures in the sample.

Gala3Gal sequences. In the mass spectrum recorded from the pooled acidicN-glycan fraction, the signals corresponding to glycan structurescontaining the Hex₆HexNAc₄ composition feature together accounted forabout 16% of the total signal intensity, which is consistent with theNMR result that these signals correspond to major glycans in the sample.

Comparison of NMR profiling and mass spectrometric profiling results. Asdescribed above, the ¹H-NMR spectra were consistent with the massspectra recorded from the hESC samples and support the quantitative andstructural assignments made based on the mass spectrometric profiles inthe preceding Examples.

NMR REFERENCES

-   Fu D., Chen L. and O'Neill R. A. (1994) Carbohydr. Res. 261, 173-186-   Helin J., Maaheimo H., Seppo A., Keane A. and Renkonen O. (1995)    Carbohydr. Res. 266, 191-209-   Hard K., Mekking A., Kamerling J. P., Dacremont G. A. A. and    Vliegenthart J. F. G. (1991) Glycoconjugate J. 8, 17-28-   H{dot over (a)}rd K., Van Zadelhoff G., Moonen P., Kamerling J. P.    and Vliegenthart J. F. G. (1992) Eur. J. Biochem. 209, 895-915

Example 14 O-glycan Profiling of Human Stem Cells Methods

Reductive β-elimination. The procedure has been described (Nyman et al.,1998). Briefly, glycoproteins were dissolved in 1 M NaBH in 0.1 M NaOHand incubated at 37° C. for two days. Borohydride was destroyed byrepeated evaporation from mild acetic acid in methanol. The resultingglycan alditols were purified by solid-phase extraction methods asdescribed above.

Non-reductive β-elimination. The procedure has been described (Huang etal., 2001). Briefly, glycoproteins were dissolved in ammonium carbonatein concentrated ammonia and incubated at 60° C. for two days. Thereagents were removed by evaporation and glycosylamines by briefincubation and evaporation from mild aqueous acetic acid. The resultingreducing glycans were purified by solid-phase extraction methods asdescribed above.

Mass spectrometry and data analysis were performed as described in thepreceding Examples.

Results and Discussion

O-glycans in cord blood mononuclear cells. O-glycan fraction wasisolated by reductive β-elimination from total glycoprotein fractions ofcord blood mononuclear cells. The glycan alditols were divided intoneutral and acidic fractions and analyzed by MALDI-TOF mass spectrometryas described above. The identified neutral and acidic glycan alditolsignals are presented in Table 19 and Table 20, respectively, and theirrelative abundances are described in FIG. 28 and FIG. 29. The glycansignals in the present example include both N- and O-glycan alditolsignals.

O-glycans in human embryonic stem cells. O-glycans were isolated bynon-reductive β-elimination from total glycoprotein fractions of humanembryonic stem cells (hESC) grown on mouse feeder cell layers. Theglycans were divided into neutral and acidic fractions and analyzed byMALDI-TOF mass spectrometry as described above. The identified glycansignals in the neutral and acidic glycans fractions are presented inTable 21 and Table 22, respectively. The most abundant potentialO-glycan signals were Hex₁HexNAc₂, Hex₂HexNAc₂, Hex₂HexNAc₂dHex₁,Hex₃HexNAc₃, Hex₃HexNAc₃dHex₁, NeuAc₂Hex₁HexNAc₁, NeuAc₁Hex₂HexNAc₂,NeuAc₁Hex₂HexNAc₂dHex₁, NeuAc₂Hex₂HexNAc₂, NeuAc₁Hex₃HexNAc₃,NeuAc₂Hex₂HexNAc₂dHex₁, NeuAc₁Hex₃HexNAc₃, Hex₃HexNAc₃SP, Hex₄HexNAc₄SP,and Hex₄HexNAc₄dHex₁SP, wherein SP corresponds to a charged group with amass of sulphate or phosphate such as sulphate ester linked to anN-acetyllactosamine structure.

Example 15 Glycosaminoglycan Fragment Analyses from Human Stem Cells

N-glycan and soluble glycan fractions were prepared from human cordblood cell populations as described in the preceding Examples. In cordblood mononuclear cells as well as affinity-purified cord blood CD34+,CD34−, CD133−, and LIN+ cell populations, following glycan fragmentswere identified (approximate experimental m/z for [M-H]⁻ ions inparenthesis): R¹ (816), R¹HexNAc₁ (1019), R² (1058), R¹HexNAc₁HexA₁(1195), R²HexA₁ (1234), R¹HexNAc₂HexA₁ (1398), R²HexNAc₁HexA₁ (1437),R¹HexNAc₂HexA₂ (1574), R²HexNAc₁HexA₂ (1613), R¹HexNAc₃HexA₂ (1777),R²HexNAc₂HexA₂ (1816), R²HexNAc₂HexA₃ (1992), and R²HexNAc₃HexA₃ (2195),wherein R¹ is preferentially HexA₁Hex₂Pen₁R³, R² is preferentiallyHexA₁Hex₃Pen₁R⁴, R³ is preferentially SO₃Ser₁ or HPO₃Ser₁, R⁴ ispreferentially (SO₃)₂Scr₁, SO₃HPO₃Ser₁, or (HPO₃)₂Ser₁. The identifiedglycans are indicated as being glycosaminoglycan fragments present instem cell and mononuclear cell populations in human cord blood.

Example 16 Exoglycosidase Analysis of Human Embryonic Stem CellsExperimental Procedures

hESC and differentiated cell samples. The human embryonic stem cell(hESC) and embryoid body (EB) samples were prepared from hESC line FES29 (Skottman et al., 2005) essentially as described in the precedingExamples, however in the present Example the hESCs were propagated onmurine fibroblast feeder cells (mEF) and the hESC samples contained somemEF cells.

Exoglycosidase digestions were performed essentially as described(Saarinen et al., 1999) and as described in the preceding Examples. Theenzymes used were α-mannosidase and β-hexosaminidase from Jack beans (C.ensiformis, Sigma, USA), β-glucosaminidase and β1,4-galactosidase fromS. pneumoniae (rec. in E. coli, Calbiochem, USA), α2,3-sialidase from S.pneumoniae (Glyko, UK), α1,3/4-fucosidase from Xanthomonas sp.(Calbiochem, USA), α1,2-fucosidase from X. manihotis (Glyko),β1,3-galactosidase (rec. in E. coli, Calbiochem), andα2,3/6/8/9-sialidase from A. ureafaciens (Glyko). The specificactivities of the enzymes were controlled in parallel reactions withpurified oligosaccharides or oligosaccharide mixtures, and analyzedsimilarly as the analytic reactions. The changes in the exoglycosidasedigestion result Tables are relative changes in the recorded massspectra and they do not reflect absolute changes in the glycan profilesresulting from glycosidase treatments.

Results and Discussion

hESC

Neutral and acidic N-glycan fractions were isolated from hESC grown onboth murine and human fibroblast feeder cells as described in thepreceding Examples. The results of parallel exoglycosidase digestions ofthe neutral (Tables 23 and 24) and acidic (Table 25) glycan fractionsare discussed below. In the following chapters, the glycan signals arereferred to by their proposed monosaccharide compositions according tothe Tables of the present invention and the corresponding m/z values canbe read from the Tables.

α-mannosidase sensitive structures. All the glycan signals that showeddecrease upon α-mannosidase digestion of the neutral N-glycan fraction(Tables 23 and 24) are indicated to correspond to glycans that containterminal α-mannose residues. The present results indicate that themajority of the neutral N-glycans of hESC contain terminal α-mannoseresidues. On the other hand, increased signals correspond to theirreaction products. Structure groups that form series of α-mannosylatedglycans in the neutral N-glycan fraction as well as individualα-mannosylated glycans are discussed below in detail.

The Hex₁₋₉HexNAc₁ glycan series was digested so that Hex₃₋₉HexNAc₁ weredigested and transformed into Hex₁HexNAc₁ (data not shown), indicatingthat they had contained terminal α-mannose residues. Because they weretransformed into Hex₁HexNAc₁, their experimental structures were(Manα)₁₋₈Hex₁HexNAc₁.

The Hex₁₋₁₂HexNAc₂ glycan series was digested so that Hex₃₋₁₂HexNAc₂were digested and transformed into Hex₁₋₇HexNAc₂ and especially intoHex₁HexNAc₂ that had not existed before the reaction and was the majorreaction product. This indicates that 1) glycans Hex₃₋₁₂HexNAc₂ includeglycans containing terminal a-mannose residues, 2) glycans Hex₃₋₇HexNAc₂could be formed from larger α-mannosylated glycans, and 3) majority ofthe glycans Hex₃₋₁₂HexNAc₂ were transformed into newly formedHex₁HexNAc₂ and therefore had the experimental structures(Manα)_(n)Hex₁HexNAc₂, wherein n≧1. The fact that the α-mannosidasereaction was only partially completed for many of the signals suggeststhat also other glycan components are included in the Hex₁₋₁₂HexNAc₂glycan series. In particular, the Hex₁₀₋₁₂HexNAc₂ components contain 1-3hexose residues more than the largest typical mammalian high-mannosetype N-glycan, suggesting that they contains glucosylated structuresincluding (Glcα)₁₋₃Hex8HexNAc₂, preferentially α2- and/or α3-linked Glcand even more preferentially present in the glucosylated N-glycansGlcα3→Man₉GlcNAc₂, Glcα2Glcα3→Man₉GlcNAc₂, and/orGlcα2Glcα2Glcα3→Man₉GlcNAc₂. The corresponding glucosylated fragmentswere observed after the α-mannosidase digestion, preferentiallycorresponding to Glc₁₋₃Man₄GlcNAc₂ (Hex₅₋₇HexNAc₂).

The Hex₁₋₆HexNAc₁dHex₁ glycan series was digested so thatHex₃₋₉HexNAcdHex₁ were digested and transformed into Hex₁HexNAc₁dHex₁,indicating that they had contained terminal α-mannose residues and theirexperimental structures were (Manα)₂₋₅Hex₁HexNAc₁dHex₁. Hex₁HexNAc₁dHex₁appeared as a new signal indicating that glycans with structures(Manα)_(n)Hex₁HexNAc₁dHex₁, wherein n≧1, had existed in the sample.

The Hex₂₋₇HexNAc₃ glycan series was digested so that Hex₅₋₇HexNAc₃ weredigested and transformed into other glycans in the series, indicatingthat they had contained terminal α-mannose residues. Hex₂HexNAc₃appeared as a new signal indicating that glycans with structures(Manα)_(n)Hex₂HexNAc₃, wherein n≧1, had existed in the sample.

The Hex₂₋₇HexNAc₃dHex₁ glycan series was digested so thatHex₅₋₇HexNAc₃dHex₁ were digested and transformed into other glycans inthe series, indicating that they had contained terminal α-mannoseresidues. Hex₂HexNAc₃dHex₁ was increased significantly indicating thatglycans with structures (Manα)_(n)Hex₂HexNAc₃dHex₁, wherein n≧1, hadexisted in the sample.

Hex₃HexNAc₃dHex₂ appeared as a new signal indicating that glycans withstructures (Manα)_(n)Hex₃HexNAc₃dHex₂, wherein n≧1, had existed in thesample.

β-glucosaminidase sensitive structures. The Hex₃HexNAC₂₋₅ andHex₃HexNAc₂₋₅dHex₁-glycan series were digested so thatHex₃₋₅HexNAc₁dHex₀₋₁ were digested and transformed intoHex₃HexNAc₂dHex₀₋₁, indicating that they had contained terminal β-GlcNAcresidues and their experimental structures were (GlcNAcβ→)₁₋₃Hex₃HexNAc₂and (GlcNAcβ→)₁₋₃Hex₃HexNAc₂dHex₁, respectively.

Hex₄HexNAc₄, Hex₄HexNAc₄dHex₁, Hex₄HexNAc₄dHex₂, and Hex₅HexNAc₅dHex₁were also digested indicating they contained structures including(GlcNAcβ→)Hex₄HexNAc₃, (GlcNAcβ→)Hex₄HexNAc₃dHex₁,(GlcNAcβ→)Hex₄HexNAc₃dHex₂, and (GlcNAcβ→)Hex₅HexNAc₄dHex₁,respectively.

Hex₄HexNAc₅dHex₁ and Hex₄HexNAc₅dHex₂ were digested by β-glucosaminidaseand indicated to contain two β-GlcNAc residues each. In contrast,Hex₄HexNAc₅ was not digested with β-glucosaminidase.

β-hexosaminidase sensitive structures. The Hex₄HexNAc₅ glycan signal wassensitive to β-hexosaminidase but not to β-glucosaminidase indicatingthat it corresponded to glycan structures containing terminalβ-N-acetylhexosamine residues other than β-GlcNAc, preferentiallyβ-GalNAc. Upon β-hexosaminidase digestion, the signal was transformedinto, Hex₄HexNAc₃ indicating that the enzyme liberated two HexNAcresidues from the corresponding glycan structures.

β1,4-galactosidase sensitive structures. Glycan signals that weresensitive to β1,4-galactosidase comprised a major proportion of hESCglycans, indicating that β1,4-linked galactose is a common terminalepitope in hESC neutral N-glycans.

Hex₅HexNAc₄ and Hex₅HexNAc₄dHex₁ were digested into Hex₃HexNAc₄ andHex₃HexNAc₄dHex₁ indicating they had the structures(Galβ4GlcNAcβ→)₂Hex₃HexNAc₂ and (Galβ4GlcNAcβ→)₂Hex₃HexNAc₂dHex₁,respectively. In contrast, Hex₅HexNAc₄dHex₂ was digested intoHex₄HexNAc₄dHex₂ indicating that it had the structure(Galβ4GlcNAcβ→)Hex₄HexNAc₃dHex₂, and Hex₅HexNAc₄dHex₃ was not digestedat all. Taken together, in hESC, hexose residues are protected bydeoxyhexose residues from the action of β1,4-galactosidase in theN-glycan structures. Such dHex-protected structures containingβ1,4-linked galactose include Galβ4(Fucα3)GlcNAc and Fucα2Galβ4GlcNAc.

Hex₄HexNA₅ that also included a β-hexosaminidase sensitive component wasdigested by β1,4-galactosidase. Taken together, the results suggest thatthe Hex₄HexNAc₅ glycan signal includes glycan structures includingGalβ4GlcNAc(GalNAcβHexNAcβ)Hex₃HexNAc₂.

β1,3-galactosidase sensitive structures. Because only few structures inhESC neutral N-glycan fraction were sensitive to the action ofβ1,3-galactosidase, the majority of terminal galactose residues appearto be β1,4-linked.

Glycosidase resistant structures. In the present experiments,Hex₄HexNAc₃, Hex₄HexNAc₃dHex₂, and Hex₅HexNAc₅ were resistant to thetested exoglycosidases. The second monosaccharide composition containsmore than one deoxyhexose residues suggesting that it is protected fromglycosidase digestions by dHex residues such as α2-, α3-, or α4-linkedfucose residues, preferentially present in Fucα2Gal, Fucα3GlcNAc, and/orFucα4GlcNAc epitopes.

The compiled neutral N-glycan fraction glycan structures based on theexoglycosidase digestions of hESC are presented in Table 26.

Acidic N-glycan fraction. The acidic N-glycan fraction of hESC grown onmEF cell layers were characterized by parallel α2,3-sialidase and A.ureafaciens sialidase treatments as well as sequential digestions withα1,3/4-fucosidase and α1,2-fucosidase. The results from these reactionsas analyzed by MALDI-TOF mass spectrometry are described in Table 25.The results suggest that multiple N-glycan components in the hESC samplecontain the specific glycan substrates for these enzymes, namelyα2,3-linked and other sialic acid residues, and both α1,2- andα1,3/4-linked fucose residues. Some glycan signals showed the presenceof many of these epitopes, such as the glycan signal at m/z 2222(corresponding to NeuAc₁Hex₅HexNAc₄dHex₂) that was suggested to containall these epitopes, preferentially in multiple glycan structures. Thecompiled acidic N-glycan fraction glycan structures based on theexoglycosidase digestions of hESC are presented in Table 27.

EB

Differentiation specific changes between embryoid bodies (EB; FES 29 st2 in Table 23) and hESC (FES 29 st 1 in Table 23) were reflected intheir neutral N-glycan fraction exoglycosidase digestion profiles, asdescribed in Table 23. Differential exoglycosidase digestion resultswere observed in glycan signals including m/z 1688, 1704, 1793, 1866,1955, 1971, 2012, 2028, 2142, 2158, and 2320, corresponding to differentneutral N-glycan fraction glycan profiles.

meF

By comparison of Table 22 and Table 23, murine feeder cell (mEF)specific neutral N-glycan fraction glycan components were identified andthey are listed in Table 28. These glycan components are characterizedby additional hexose residues compared to hESC or hEF specificstructures according to the present invention. The exoglycosidaseexperiments also suggest that β1,4-linked galactose epitopes areprotected from β1,4-galactosidase digestion by any additional hexoseresidues in the monosaccharide compositions. Taken together with the NMRanalysis results of the present invention, the additional hexoseresidues are suggested to be α-linked galactose residues, morespecifically including Galα3Gal epitopes in the N-glycan antennae, asdescribed in Table 28.

Example 17 Exoglycosidase Analysis of Human Mesenchymal Stem cells

The changes in the exoglycosidase digestion result Tables are relativechanges in the recorded mass spectra and they do not reflect absolutechanges in the glycan profiles resulting from glycosidase treatments.The experimental procedures are described in the preceding Example.

Results Undifferentiated BM MSC

Neutral and acidic N-glycan fractions were isolated from BM MSC asdescribed. The results of parallel exoglycosidase digestions of theneutral (Table 29) and acidic (data not shown) glycan fractions arediscussed below. In the following chapters, the glycan signals arereferred to by their proposed monosaccharide compositions according tothe Tables of the present invention and the corresponding m/z values canbe read from the Tables.

α-mannosidase sensitive structures. All the glycan signals that showeddecrease upon α-mannosidase digestion of the neutral N-glycan fraction(Table 29) are indicated to correspond to glycans that contain terminalα-mannose residues. The present results indicate that the majority ofthe neutral N-glycans of BM MSC contain terminal α-mannose residues. Onthe other hand, increased signals correspond to their reaction products.Structure groups that form series of α-mannosylated glycans in theneutral N-glycan fraction as well as individual α-mannosylated glycansare discussed below in detail.

The Hex₁₋₉HexNAc₁ glycan series was digested so that Hex₃₋₉HexNAc₁ weredigested and transformed into Hex₁HexNAc₁ (data not shown), indicatingthat they had contained terminal α-mannose residues. Because they weretransformed into Hex₁HexNAc₁, their experimental structures were(Manα)₁₋₈Hex₁HexNAc₁.

The Hex₁₋₁₀HexNAc₂ glycan series was digested so that Hex₄₋₁₀HexNAc₂were digested and transformed into Hex₁HexNAc₂ and especially intoHex₁HexNAC₂ that had not existed before the reaction and was the majorreaction product. This indicates that 1) glycans Hex₄₋₁₀HexNAc₂ includeglycans containing terminal α-mannose residues, 2) glycans Hex₁₋₄HexNAc₂could be formed from larger α-mannosylated glycans, and 3) majority ofthe glycans Hex₄₋₁₀HexNAc₂ were transformed into newly formedHex₁HexNAc₂ and therefore had the experimental structures(Manα)_(n)Hex₁HexNAc₂, wherein n≧1. The fact that the α-mannosidasereaction was only partially completed for many of the signals suggeststhat also other glycan components are included in the Hex₁₋₁₀HexNAc₂glycan series. In particular, the Hex₁₀HexNAc₂ component contains onehexose residue more than the largest typical mammalian high-mannose typeN-glycan, suggesting that it contains glucosylated structures including(Glcα→)Hex₈HexNAc₂, preferentially α3-linked Glc and even morepreferentially present in the glucosylated N-glycan (Glcα3→)Man₉GlcNAc₂.

The Hex₁₋₆HexNAc₁dHex₁ glycan series was digested so thatHex₃₋₉HexNAc₁dHex₁ were digested and transformed into Hex₁HexNAc₁dHex₁,indicating that they had contained terminal α-mannose residues and theirexperimental structures were (Manα)₂₋₅Hex₁HexNAc₁dHex₁. Hex₁HexNAc₁dHex₁appeared as a new signal indicating that glycans with structures(Manα)_(n)Hex₁HexNAc₁dHex₁, wherein n≧1, had existed in the sample.

The Hex₂₋₇HexNAc₃ glycan series was digested so that Hex₆₋₇HexNAc₃ weredigested and transformed into other glycans in the series, indicatingthat they had contained terminal α-mannose residues. Hex₂HexNAc₃appeared as a new signal indicating that glycans with structures(Manα)_(n)Hex₂HexNAc₃, wherein n≧1, had existed in the sample.

The Hex₂₋₇HexNAc₃dHex₁ glycan series was digested so thatHex₆₋₇HexNAc₃dHex₁ were digested and transformed into other glycans inthe series, indicating that they had contained terminal α-mannoseresidues. Hex₂HexNAc₃dHex₁ appeared as a new signal indicating thatglycans with structures (Manα)_(n)Hex₂HexNAc₃dHex₁, wherein n≧1, hadexisted in the sample.

Hex₃HexNAc₃dHex₂ and Hex₃HexNAc₄ appeared as new signals indicating thatglycans with structures (Manα)_(n)Hex₃HexNAc₃dHex₂ and(Manα)_(n)Hex₃HexNAc₄, respectively, wherein n≧1, had existed in thesample.

β-glucosaminidase sensitive structures. The Hex₃HexNAc₂₋₅dHex₁ glycanseries was digested so that Hex₃₋₉HexNAc₁dHex₁ were digested andtransformed into Hex₁HexNAc₁dHex₁, indicating that they had containedterminal α-mannose residues and their experimental structures were(Manα)₂₋₅Hex₁HexNAc₁dHex₁. Hex₁HexNAc₁dHex₁, appeared as a new signalindicating that glycans with structures (Manα)_(n)Hex₁HexNAc₁dHex₁,wherein n≧1, had existed in the sample. However, Hex₃HexNAc₆dHex₁ wasnot digested indicating that it contained other terminal HexNAc residuesthan β-linked GlcNAc residues.

Hex₂HexNAc₃ and Hex₂HexNAc₃dHex₁ were digested into Hex₂HexNAc₂ andHex₂HexNAc₂dHex₁ indicating they had the structures(GlcNAcβ→)Hex₂HexNAc₂ and (GlcNAcβ→)Hex₂HexNAc₂dHex₁, respectively.

Hex₄HcxNAc₄dHex₁, Hex₄HexNAc₄dHex₂, Hex₄HexNAc₅dHex₂, andHex₅HexNAc₅dHex₁ were also digested indicating they contained structuresincluding (GlcNAcβ→)Hex₄HexNAc₃dHex₁, (GlcNAcβ→)Hex₄HexNAc₃dHex₂,(GlcNAcβ→)Hex₄HexNAc₄dHex₂, and (GlcNAcβ→)Hex₅HexNAc₄dHex₁,respectively.

β1,4-galactosidase sensitive structures. Glycan signals that weresensitive to β1,4-galactosidase comprised a major proportion of BM MSCglycans, indicating that β1,4-linked galactose is a common terminalepitope in BM MSC neutral N-glycans.

Hex₅HexNAc₄ and Hex₅HexNAc₄dHex₁ were digested into Hex₃HexNAc₄ andHex₃HexNAc₄dHex₁ indicating they had the structures(Galβ4GlcNAcβ→)₂Hex₃HexNAc₂ and (Galβ4GlcNAcβ→)₂Hex₃HexNAc₂dHex₁,respectively. In contrast, Hex₅HexNAc₄dHex₂ was digested intoHex₄HexNAc₄dHex₂ indicating that it had the structure(Galβ4GlcNAcβ→)Hex₄HexNAc₃dHex₂, respectively, and Hex₅HexNAc₄dHex₃ wasnot digested at all. Taken together, in BM MSC, n−1 hexose residues areprotected by deoxyhexose residues from the action of β1,4-galactosidasein the N-glycan structures Hex₅HexNAc₄dHex_(n), wherein 0≦n≦3. SuchdHex-protected structures containing β1,4-linked galactose includeGalβ4(Fucα3)GlcNAc and Fucα2Galβ4GlcNAc.

Similarly, Hex₆HexNAc₅, Hex₅HexNAc₅dHex₁, Hex₆HexNAc₅, andHex₅HexNAc₅dHex₁ were digested into Hex₃HexNAc₅, Hex₃HexNAc₅dHex₁, andHex₃HexNAc₆dHex₁ ndicating they had the structures(Galβ4GlcNAcβ→)₃Hex₃HexNAc₂, (Galβ4GlcNAcβ→)₂Hex₃HexNAc₃dHex₁, and(Galβ4GlcNAcβ→)₃Hex₃HexNAc₃dHex₁, respectively. In contrast,Hex₄HexNAc₅dHex₂, Hex₅HexNAc₅dHex₃, Hex₆HexNAc₅dHex₂, andHex₆HexNAc₅dHex₃ were not digested, indicating that hexose residues inthese structures were protected by deoxyhexose residues. SuchdHex-protected structures containing β1,4-linked galactose includeGalβ4(Fucα3)GlcNAc and Fucα2Galβ4GlcNAc. However, Hex₄HexNAc₅dHex₃ wasdigested indicating that it contained one or more terminal β1,4-linkedgalactose residues.

Hex₇HexNAc₃, Hex₆HexNAc₃dHex₁, Hex₆HexNAc₃, and HeX₅HexNAc₃dHex₁ weredigested into products including Hex₅HexNAc₃ and Hex₄HexNAc₃dHex₁,indicating they had the structures (Galβ4GlcNAcβ→)Hex₅HexNAc₂ and(Galβ4GlcNAcβ→)Hex₄₋₅HexNAc₃dHex₁, respectively. The relative amounts ofHex₃HexNAc₃, and Hex₃HexNAc₃dHex₁ were increased indicating that theywere products of (Galβ4GlcNAcβ→)Hex₃HexNAc₂ and(Galβ4GlcNAcβ→)Hex₃HexNAc₂dHex₁, respectively.

β1,3-galactosidase sensitive structures. Because only few structures inBM MSC neutral N-glycan fraction are sensitive to the action ofβ1,3-galactosidase, the majority of terminal galactose residues appearto be β1,4-linked. The glycan signals corresponding toβ1,3-galactosidase sensitive glycans include Hex₅HexNAc₅dHex₁ andHex₄HexNAc₅dHex₃.

Glycosidase resistant structures. In the present experiments,Hex₂HexNAc₃dHex₂, Hex₄HexNAc₃dHex₂, and Hex₁₁HexNAc₂ were resistant tothe tested exoglycosidases. The first two proposed monosaccharidecompositions contain more than one deoxyhexose residues suggesting thatthey are protected from glycosidase digestions by the second dHexresidues such as α2-, α3-, or α4-linked fucose residues, preferentiallypresent in Fucα2Gal, Fucα3GlcNAc, and/or Fucα4GlcNAc epitopes. The lastproposed monosaccharide composition contains two hexose residues morethan the largest typical mammalian high-mannose type N-glycan,suggesting that it contains glucosylated structures including(Glcα→)₂Hex₉HexNAc₂, preferentially α2- and/or α3-linked Glc and evenmore preferentially present in the diglucosylated N-glycan(GlcαGlcα→)Man₉GlcNAc₂.

The compiled neutral N-glycan fraction glycan structures based on theexoglycosidase digestions of BM MSC are presented in Table 30.

Osteoblast-differentiated BM MSC

The analysis of osteoblast differentiated BM MSC are presented in Table31, allowing comparison of differentiation specific changes in CB MSC.The exoglycosidase profiles produced for BM MSC and osteoblastdifferentiated BM MSC are characteristic for the two cell types. Forexample, signals at m/z 1339, 1784, and 2466 are digested differentiallyin the two experiments. Specifically, the presence of β1,3-galactosidasesensitive neutral N-glycan signals in osteoblast differentiated BM MSCindicate that the differentiated cells contain more β1,3-linkedgalactose residues than the undifferentiated cells.

The sialidase analysis performed for the acidic N-glycan fraction of BMMSC supported the proposed monosaccharide compositions based onsialylated (NeuAc or NeuGc containing) N-glycans in the acidic N-glycanfraction.

Analysis of CB MSC Neutral Glycan Fraction by Exoglycosidases

The results of the analysis by β1,4-galactosidase and 0-glucosaminidaseare presented in Table 32. The results suggest that also in CB MSCneutral N-glycans containing non-reducing terminal β1,4-linked galactoseresidues are abundant, and they suggest the presence of characteristicnon-reducing terminal epitopes for most of the observed glycan signals.The analysis of adipocyte differentiated CB MSC are presented in Table33, allowing comparison of differentiation specific changes in CB MSC,similarly as described above for BM MSC.

The sialidase analysis performed for the acidic N-glycan fraction of CBMSC supported the proposed monosaccharide compositions based onsialylated (NeuAc or NeuGc containing) N-glycans in the acidic N-glycanfraction.

Example 18 Analysis of Acidic Glycans Results and Discussion

Acidic glycans containing sulphate or phosphate ester groups. The celltype specific occurrence of glycan signals corresponding tomonosaccharide compositions containing sulphate or phosphate estergroups are listed in Table 46.

Acidic glycans containing sialidase-resistant sulphate or phosphateester groups. The glycan signals in hESC and CB MNC corresponding tomonosaccharide compositions containing sulphate or phosphate estergroups (SP) were studied by treating the acidic N-glycan fractionsisolated from these cells by A. ureafaciens sialidase as describedabove, and analyzing the sialidase-resistant glycan signals after thetreatment as described above. In both these cell types, specific glycansignals had resisted the action of sialidase and were assigned either asnative SP-containing glycan signals or desialylated SP-containing glycansignals. Such signals are indicated for hESC in Table 26 as signalscontaining SP in their monosaccharide compositions (marked with +, ++,or +++ in Table 26), and selected in a separate table (Table 34) for CBMNC.

Fragmentation mass spectrometry of stem cell N-glycans. Acidic N-glycansisolated from a bone marrow derived mesenchymal stem cell line wereanalyzed by MALDI-TOF mass spectrometry in negative ion mode. Thespectrum showed the presence of glycan signals containing sulphate orphosphate ester (SP) in their proposed monosaccharide compositions, asdescribed in the Tables of the present invention. One such glycan signalwas at m/z 1719, corresponding to the [M-H]⁻ ion of Hex₅HexNAc₄SP₁. Whenthe same sample was analyzed by MALDI-TOF mass spectrometry in positiveion mode, a corresponding signal was detected at m/z 1765 for the ion[M-H+2Na]⁺, but not at m/z 1743 for the ion [M+Na]⁺, suggesting that themolecule contained an acidic group that was ionized and present assodium salt in positive ion mode mass spectrometry. When the ion at m/z1765 was subjected to fragmentation, a fragmentation mass spectrum inFIG. 30 was recorded. The fragmentation spectrum showed the majorfragment at m/z 1663 corresponding to the [M+Na]⁺ ion of Hex₅HexNAc₄(resulting from elimination of SPNa, sodium salt of sulphate orphosphate ester). However, no fragmentation products were observed atm/z 1452 that would have corresponded to elimination of sialic acid fromthe parent ion. Taken together, the results of the fragmentationexperiment supported the presence of sulphate or phosphate ester in theglycan signal at m/z 1719 in the negative ion mode mass spectrum and atm/z 1765 in the positive ion mode mass spectrum. The observed fragmentions and their proposed monosaccharide compositions were: m/z 1765.75,[M-H+2Na]⁺/Hex₅HexNAc₄SP₁ (parent ion); m/z 1663.22,[M+Na]⁺/Hex₅HexNAc₄; m/z 1605.45, unidentified fragment; m/z 1544.52,[M-H+2Na—H₂O]⁺/Hex₅HexNAc₃SP₁-H₂O; m/z 1475.34, unidentified fragment;m/z 1459.92, [M+Na]⁺/Hex₅HexNAc₃; m/z 1444.18,[M-H+2Na—H₂O]⁺/Hex₅HexNAc₃-H₂O; m/z 1400.35, [M-H+2Na]⁺/Hex₄HexNAc₃SP₁;m/z 1539.23, [M-H+2Na]⁺/Hex₅HexNAc₂SP₁; m/z 1341.17,[M-H+2Na—H₂O]⁺/Hex₅HexNAc₂SP₁-H₂O; m/z 1298.26, [M+Na]⁺/Hex₄HexNAc₃.

Fragmentation mass spectrometry of mouse fibroblast feeder cellN-glycans. Acidic N-glycans isolated from a mouse fibroblast feeder cellline were analyzed by MALDI-TOF mass spectrometry in negative ion mode.The spectrum showed the presence of glycan signals containing anadditional hexose in their proposed monosaccharide compositions(n_(Hex)=n_(HexNAc)+2), as described in the preceding Examples. One suchglycan signal was at m/z 2238, corresponding to the [M-H]⁻ ion ofNeuAc₁Hex₆HexNAc₄dHex₁. When the same sample was analyzed by MALDI-TOFmass spectrometry in positive ion mode, a corresponding signal wasdetected at m/z 2284 for the ion [M-H+2Na]⁺. When glycans at m/z 2284were subjected to fragmentation (data not shown), the fragmentationspectrum showed the major fragment at m/z 1971.30 corresponding to the[M+Na]⁺ ion of Hex₆HexNAc₄dHex₁ (resulting from elimination of NeuAcNa,or sodium salt of an acetylneuraminic acid residue). Other observedfragment ions and their proposed monosaccharide compositions were: m/z2122.12 corresponding to the [M-H+2Na]⁺ ion of NeuAc₁Hex₅HexNAc₄dHex₁,m/z 1808.96 corresponding to the [M+Na]⁺ ion of Hex₅HexNAc4dHex₁, andm/z 1606.23 corresponding to the [M+Na]⁺ ion of Hex₅HexNAc₃dHex₁.

Example 19 Lectin and Antibody Profiling of Human Embryonic Stem CellsExperimental Procedures

Cell samples. Human embryonic stem cell (hESC) lines FES 22 and FES 30(Family Federation of Finland) were propagated on mouse feeder cell(mEF) layers as described above.

FITC-labeled lectins. Fluorescein isotiocyanate (FITC) labeled lectinswere purchased from several manufacturers: FITC-GNA, -HRA, -MAA, -PWA,-STA and -LTA were from EY Laboratories (USA); FITC-PSA and -UEA andbiotin-labelled WFA were from Sigma (USA); and FITC-RCA, -PNA and -SNAwere from Vector Laboratories (UK).

Fluorescence microscopy labeling experiments were conducted essentiallyas described in the preceding Examples. Biotin label was visualized byfluorescein-conjugated streptavidin.

Results

Table 35 shows the tested FITC-labelled lectins, examples of theirtarget saccharide sequences, and the graded lectin binding intensitiesas described in the Table legend, in fluorescence microscopy of fixedcells grown on microscopy slides. Multiple binding specificities for theused lectins are described in the art and in general the binding of alectin in the present experiments means that the cells express specificligands for the lectin on their surface, but does not exclude thepresence of also other ligands that are recognized by the lectin.

α-linked mannose. Abundant labelling of mEF by Pisum sativum (PSA)lectins suggests that they express mannose, more specifically α-linkedmannose residues on their surface glycoconjugates such as N-glycans. Theresults further suggest that the both hESC lines do not express theseligands at as high concentrations as mEF on their surface.

β-linked galactose. Abundant labelling of hESC by peanut lectin (PNA)and less intense labelling by Ricinus communis lectin I (RCA-I) suggeststhat hESC express β-linked non-reducing terminal galactose residues ontheir surface glycoconjugates such as N- and/or O-glycans. Morespecifically, RCA-I binding suggests that the cells contain high amountsof unsubstituted Galβ epitopes on their surface. PNA binding suggestsfor the presence of unsubstituted Galβ, and the absence of specificbinding of PNA to mEF suggests that the binding epitopes for this lectinare less abundant in mEF.

Sialic acids. Specific labelling of hESC by both Maackia amurensis (MAA)and Sambucus nigra (SNA) lectins suggests that the cells express sialicacid residues on their surface glycoconjugates such as N- and/orO-glycans and/or glycolipids. More specifically, the specific MAAbinding of hESC suggests that the cells contain high amounts ofα2,3-linked sialic acid residues. In contrast, the results suggest thatthese epitopes are less abundant in mEF. SNA binding in both cell typessuggests for the presence of also α2,6-linkages in the sialic acidresidues on the cell surface.

Poly-N-acetyllactosamine sequences. Labelling of the cells by pokeweed(PWA) and less intense labelling by Solanum tuberosum (STA) lectinssuggests that the cells express poly-N-acetyllactosamine sequences ontheir surface glycoconjugates such as N- and/or O-glycans and/orglycolipids. The results further suggest that cell surfacepoly-N-acetyllactosamine chains contain both linear and branchedsequences.

β-linked N-acetylgalactosamine. Abundant labelling of hESC by Wisteriafloribunda lectin (WFA) suggests that hESC express β-linked non-reducingterminal N-acetylgalactosamine residues on their surface glycoconjugatessuch as N- and/or O-glycans. The absence of specific binding of WFA tomEF suggests that the lectin ligand epitopes are less abundant in mEF.

Fucosylation. Labelling of the cells by Ulex europaeus (UEA) and lessintense labelling by Lotus tetragonolobus (LTA) lectins suggests thatthe cells express fucose residues on their surface glycoconjugates suchas N- and/or O-glycans and/or glycolipids. More specifically, the UEAbinding suggests that the cells contain α-linked fucose residuesincluding α1,2-linked fucose residues. LTA binding suggests for thepresence of α-linked fucose residues including α1,3- or α1,4-linkedfucose residues on the cell surface.

The specific antibody anti-Lex and anti-sLex antibody binding resultsindicate that the hESC samples contain Galβ4(Fucα3)GlcNAcβ andSAα3Galβ4(Fucα3)GlcNAcβ carbohydrate epitopes on their surface,respectively.

Taken together, in the present experiments the lectins PNA, MAA, and WFAas well as the antibodies anti-Lex and anti-sLex bound specifically tohESC but not to mEF. In contrast, the lectin PSA bound specifically tomEF but not to hESC. This suggests that the glycan epitopes that thesereagents recognize have hESC or mEF specific expression patterns. On theother hand, other reagents in the tested reagent panel bounddifferentially to the two hESC lines FES 22 and FES 30, indicating cellline specific glycosylation of the hESC cell surfaces (Table 35).

Discussion

Venable, A., et al. (2005 BMC Dev. Biol.) have previously describedlectin binding profiles of SSEA-4 enriched human embryonic stem cells(hESC) grown on mouse feeder cells. The lectins used were Lycopersiconesculentum (LEA, TL), RCA, Concanavalin A (ConA), WFA, PNA, SNA,Hippeastrum hybrid (HHA, HHL), Vicia villosa (VVA), UEA, Phaseolusvulgaris (PHA-L and PHA-E), MAA, LTA (LTL), and Dolichos biflorus (DBA)lectins. In FACS and cytochemistry analysis, four lectins were found tohave similar binding percentage as SSEA-4 (LEA, RCA, ConA, and WFA) andin addition two lectins also had high binding percentage (PNA and SNA).Two lectins did not bind to hESCs (DBA and LTA). Six lectins were foundto partially bind to hESC(PHA-E, VVA, UEA, PHA-L, MAA, and HHA). Theauthors suggested that the differential lectin binding specificities canbe used to distinguish hESC and differentiated hESC types based oncarbohydrate presentation.

Venable et al. (2005) discuss some carbohydrate structures that theyclaim to have high expression on the surface of pluripotent SSEA-4 hESC(corresponding lectins according to Venable et al. in parenthesis):α-Man (ConA, HHA), Glc (ConA), Galβ3GalNAcβ(PNA), non-reducing terminalGal (RCA), non-reducing terminal β-GalNAc (RCA), GalNAcp4Gal (WFA),GlcNAc (LEA), and SAα6GalNAc (SNA). In addition, Venable et al., discusssome carbohydrate structures that they claim to have expression onsurface of a proportion of pluripotent SSEA-4 hESC (correspondinglectins according to Venable et al. in parenthesis): Gal (PHA-L, PHA-E,MAA), GalNAc (VVA) and Fuc (UEA). However, ConA is not especiallyspecific to Glc and MAA has no specificity to Gal residues.

In the present experiments, RCA binding was observed on both hESC lineFES 22 and mEF, but not on FES 30. This suggests that RCA bindingspecificity in hESC varies from cell line to another. The presentexperiments also show other lectins to be expressed on only one out ofthe two hESC lines (Table 35), suggesting that there is individualvariation in binding of some lectins.

Based on LTA not binding to hESC in their experiments, Venable et al.(2005) suggest that on hESC surface there are no non-modified fucoseresidues that are α-linked to GlcNAc. However, in the presentexperiments LTA as well as anti-Lex and anti-sLex monoclonal antibodieswere found to bind to the hESC line FES 22. The present antibody bindingresults indicate that FucαGlcNAc epitopes, specificallyGalβ4(Fucα3)GlcNAc sequences, are present on hESC surface.

Venable et al. (2005) describe that PNA recognizes in their hESC samplesspecifically Galβ3GalNAc structures, wherein the GalNAc residue isβ-linked. In the present experiments, PNA was used to recognizecarbohydrate structures generally including β-linked galactose residuesand without β-linkage requirement for the GalNAc residue.

Venable et al. (2005) describe that SNA recognizes in their hESC samplesspecifically SAα6GalNAc structures. In the present experiments, SNA wasused to recognize α2,6-linked sialic acids in general and its ligandswere also found on mEF.

Inhibition of MAA binding by 200 mM lactose in the experiments describedby Venable et al. (2005) suggests non-specific binding of MAA withrespect to sialic acids. According to the present experiments, MAA canrecognize α2,3-linked sialic acid residues on hESC surface anddifferentiate between hESC and mEF.

Example 20 Lectin and Antibody Profiling of Human Mesenchymal Stem CellsExperimental Procedures

Cell samples. Bone marrow derived human mesenchymal stem cell lines(MSC) were generated and cultured in proliferation medium as describedabove.

FITC-labeled lectins. Fluorescein isotiocyanate (FITC) labelled lectinswere purchased from several manufacturers: FITC-GNA, -HHA, -MAA, -PWA,-STA and -LTA were from EY Laboratories (USA); FITC-PSA and -UEA werefrom Sigma (USA); and FITC-RCA, -PNA and -SNA were from VectorLaboratories (UK). Lectins were used in dilution of 5 μg/10⁵ cells in 1%human serum albumin (HSA; FRC Blood Service, Finland) in phosphatebuffered saline (PBS).

Flow cytometry. Flow cytometric analysis of lectin binding was used tostudy the cell surface carbohydrate expression of MSC. 90% confluent MSClayers on passages 9-11 were washed with PBS and harvested into singlecell suspensions by 0.25% trypsin-1 mM EDTA solution (Gibco). Detachedcells were centrifuged at 600 g for five minutes at room temperature.Cell pellet was washed twice with 1% HSA-PBS, centrifuged at 600 g andresuspended in 1% HSA-PBS. Cells were placed in conical tubes inaliquots of 70000-83000 cells each. Cell aliquots were incubated withone of the FITC labelled lectin for 20 minutes at room temperature.After incubation cells were washed with 1% HSA-PBS, centrifuged andresuspended in 1% HSA-PBS. Untreated cells were used as controls. Lectinbinding was detected by flow cytometry (FACSCalibur, Becton Dickinson).Data analysis was made with Windows Multi Document Interface for FlowCytometry (WinMDI 2.8). Two independent experiments were carried out.

Fluorescence microscopy labeling experiments were conducted as describedin the preceding Examples.

Results and Discussion

Table 36 shows the tested FITC-labelled lectins, examples of theirtarget saccharide sequences, and the amount of cells showing positivelectin binding (%) in FACS analysis after mild trypsin treatment. Table37 shows the tested FITC-labelled lectins, examples of their targetsaccharide sequences, and the graded lectin binding intensities asdescribed in the Table legend, in fluorescence microscopy of fixed cellsgrown on microscopy slides. Binding specificities of the used lectinsare described in the art and in general the binding of a lectin in thepresent experiments means that the cells express specific ligands forthe lectin on their surface. The examples of some of the specificitiesdiscussed below and those marked in the Tables are thereforenon-exclusive in nature.

α-linked mannose. Abundant labelling of the cells by both Hippeastrumhybrid (RHA) and Pisum sativum (PSA) lectins suggests that they expressmannose, more specifically α-linked mannose residues on their surfaceglycoconjugates such as N-glycans. Possible α-mannose linkages includeα1→2, α1→3, and α1→6. The lower binding of Galanthus nivalis (GNA)lectin suggests that some α-mannose linkages on the cell surface aremore prevalent than others.

β-linked galactose. Abundant labelling of the cells by Ricinus communislectin I (RCA-I) and less intense labelling by peanut lectin (PNA)suggests that the cells express β-linked non-reducing terminal galactoseresidues on their surface glycoconjugates such as N- and/or O-glycans.More specifically, the intense RCA-I binding suggests that the cellscontain high amounts of unsubstituted Galp epitopes on their surface.The binding of RCA-I was increased by sialidase treatment of the cellsbefore lectin binding, indicating that the ligands of RCA-I on MSC wereoriginally partly covered by sialic acid residues. PNA binding suggestsfor the presence of another type of unsubstituted Galβ epitopes such asCore 1 O-glycan epitopes on the cell surface. The binding of PNA wasalso increased by sialidase treatment of the cells before lectinbinding, indicating that the ligands of PNA on MSC were originallymostly covered by sialic acid residues. These results suggest that bothRCA-I and PNA can be used to assess the amount of their specific ligandson the cell surface of BM MSC, and with or without conjunction withsialidase treatment to assess the sialylation level of their specificepitopes.

Sialic acids. Abundant labelling of the cells by Maackia amurensis (MAA)and less intense labelling by Sambucus nigra (SNA) lectins suggests thatthe cells express sialic acid residues on their surface glycoconjugatessuch as N- and/or O-glycans and/or glycolipids. More specifically, theintense MAA binding suggests that the cells contain high amounts ofα2,3-linked sialic acid residues on their surface. SNA binding suggestsfor the presence of also α2,6-linked sialic acid residues on the cellsurface, however in lower amounts than α2,3-linked sialic acids. Both ofthese lectin binding activities could be reduced by sialidase treatment,indicating that the specificities of the lectins in BM MSC are mostlytargeted to sialic acids.

Poly-N-acetyllactosamine sequences. Labelling of the cells by Solanumtuberosum (STA) and less intense labelling by pokeweed (PWA) lectinssuggests that the cells express poly-N-acetyllactosamine sequences ontheir surface glycoconjugates such as N- and/or O-glycans and/orglycolipids. Higher intensity labelling with STA than with PWA suggeststhat most of the cell surface poly-N-acetyllactosamine sequences arelinear and not branched or substituted chains.

Fucosylation. Labelling of the cells by Ulex europaeus (UEA) and lessintense labelling by Lotus tetragonolobus (LTA) lectins suggests thatthe cells express ficose residues on their surface glycoconjugates suchas N- and/or O-glycans and/or glycolipids. More specifically, the UEAbinding suggests that the cells contain α-linked fucose residues,including α1,2-linked fucose residues, on their surface. LTA bindingsuggests for the presence of also α-linked fucose residues, includingα1,3-linked fucose residues on the cell surface, however in loweramounts than UEA ligand fucose residues.

Mannose-binding lectin labelling. Low labelling intensity was alsodetected with human serum mannose-binding lectin (MBL) coupled tofluorescein label, suggesting that ligands for this innate immunitysystem component may be expressed on in vitro cultured BM MSC cellsurface.

Binding of a NeuGc polymeric probe (Lectinity Ltd., Russia) to non-fixedhESC indicates the presence of NeuGc-specific lectin on the cellsurfaces. In contrast, polymeric NeuAc probe did not bind to the cellswith same intensity in the present experiments.

The binding of the specific antibodies to hESC indicates the presence ofLex and sialyl-Lewis x epitopes on their surfaces, and binding ofNeuGc-specific antibody to hESC indicates the presence of NeuGc epitopeson their surfaces.

Example 21 Lectin and Antibody Profiling of Human Cord Blood CellPopulations Results and Discussion

FIG. 31 shows the results of FACS analysis of FITC-labelled lectinbinding to seven individual cord blood mononuclear cell (CB MNC)preparations (experiments performed as described above). Strong bindingwas observed in all samples by GNA, HHA, PSA, MAA, STA, and UEAFITC-labelled lectins, indicating the presence of their specific ligandstructures on the CB MNC cell surfaces. Also mediocre binding (PWA),variable binding between CB samples (PNA), and low binding (LTA) wasobserved, indicating that the ligands for these lectins are eithervariable or more rare on the CB MNC cell surfaces as the lectins above.

Example 22 Analysis of Total N-glycomes of Human Stem Cells and CellPopulations Experimental Procedures

Cell and glycan samples were prepared as described in the precedingExamples.

Relative proportions of neutral and acidic N-glycan fractions werestudied by desialylating isolated acidic glycan fraction with A.ureafaciens sialidase as described in the preceding Examples and thencombining the desialylated glycans with neutral glycans isolated fromthe same sample. Then the combined glycan fractions were analyzed bypositive ion mode MALDI-TOF mass spectrometry as described in thepreceding Examples. The proportion of sialylated N-glycans of thecombined N-glycans was calculated by calculating the percentual decreasein the relative intensity of neutral N-glycans in the combined N-glycanfraction compared to the original neutral N-glycan fraction, accordingto the equation:

${{proportion} = {\frac{I^{neutral} - I^{combined}}{I^{neutral}} \times 100\%}},$

wherein I^(neutral) and I^(combined) correspond to the sum of relativeintensities of the five high-mannose type N-glycan [M+Na]⁺ ion signalsat m/z 1257, 1419, 1581, 1743, and 1905 in the neutral and combinedN-glycan fractions, respectively.

Results and Discussion

The relative proportions of acidic N-glycan fractions in studied stemcell types were as follows: in human embryonic stem cells (hESC)approximately 35% (proportion of sialylated and neutral N-glycans isapproximately 1:2), in human bone marrow derived mesenchymal stem cells(BM MSC) approximately 19% (proportion of sialylated and neutralN-glycans is approximately 1:4), in osteoblast-differentiated BM MSCapproximately 28% (proportion of sialylated and neutral N-glycans isapproximately 1:3), and in human cord blood (CB) CD133+ cellsapproximately 38%

(proportion of sialylated and neutral N-glycans is approximately 2:3).

In conclusion, BM MSC differ from hESC and CB CD133+ cells in that theycontain significantly lower amounts of sialylated N-glycans compared toneutral N-glycans. However, after osteoblast differentiation of the BMMSC the proportion of sialylated N-glycans increases.

Example 23 Analysis of the Human Embryonic Stem Cell N-glycomeExperimental Procedures

Human embryonic stem cell lines (hESC). Four Finnish hESC lines, FES 21,FES 22, FES 29, and FES 30, were used in the present study. Generationof the lines has been described (Skotaan et al., 2005, and M.M., C.O.,T.T., and T.O., manuscript submitted for publication). Two of theanalysed cell lines in the present work were initially derived andcultured on mouse embryonic fibroblast feeders, and two on humanforeskin fibroblast feeder cells. For the mass spectrometry studies allof the lines were transferred on HFF feeder cells treated withmitomycin-C (1 μg/ml, Sigma-Aldrich, USA) and cultured in serum-freemedium (Knockout™ D-MEM; Gibco® Cell culture systems, Invitrogen, UK)supplemented with 2 mM L-Glutamin/Penicillin streptomycin(Sigma-Aldrich), 20% Knockout Serum Replacement (Gibco), 1×non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco),1×ITS (Sigma-Aldrich) and 4 ng/ml bFGF (Sigma/Invitrogen). To induce theformation of embryoid bodies (EB) the hESC colonies were first allowedto grow for 10-14 days whereafter the colonies were cut in small piecesand transferred on non-adherent Petri dishes to form suspensioncultures. The formed EBs were cultured in suspension for the next 10days in standard culture medium (see above) without bFGF. For furtherdifferentiation (into stage 3 differentiated cells) EBs were transferredonto gelatin-coated (Sigma-Aldrich) adherent culture dishes in mediaconsisting of DMEM/F12 mixture (Gibco) supplemented with ITS,Fibronectin (Sigma), L-glutamine and antibiotics. The attached cellswere cultured for 10 days whereafter they were harvested. For glycananalysis, the cells were collected mechanically, washed, and storedfrozen until the analysis. In FACS analyses 70-90% of cells frommechanically isolated hESC colonies were typically Tra 1-60 and Tra 1-81positive (not shown). Cells differentiated into embryoid bodies (EB) andfurther differentiated cells grown out of the EB as monolayers (stage 3differentiated) were used for comparison against hESC. Thedifferentiation protocol favors the development of neuroepithelial cellswhile not directing the differentiation into distinct terminallydifferentiated cell types (Okabe et al., 1996). Stage 3 culturesconsisted of a heterogenous population of cells dominated byfibroblastoid and neuronal morphologies.

Glycan isolation. Asparagine-linked glycans were detached from cellularglycoproteins by F. meningosepticum N-glycosidase F digestion(Calbiochem, USA) essentially as described (Nyman et al., 1998). Thedetached glycans were divided into sialylated and non-sialylatedfractions based on the negative charge of sialic acid residues. Cellularcontaminations were removed by precipitating the glycans with 80-90%(v/v) aqueous acetone at −20° C. and extracting them with 60% (v/v)ice-cold methanol essentially as described previously (Verostek et al.,2000). The glycans were then passed in water through C₁₈ silica resin(BondElut, Varian, USA) and adsorbed to porous graphitized carbon(Carbograph, Alltech, USA) based on previous method (Davies et al.,1993). The carbon column was washed with water, then the neutral glycanswere eluted with 25% acetonitrile in water (v/v) and the sialylatedglycans with 0.05% (v/v) trifluoroacetic acid in 25% acetonitrile inwater (v/v). Both glycan fractions were additionally passed in waterthrough strong cation-exchange resin (Bio-Rad, USA) and C₁₈ silica resin(ZipTip, Millipore, USA). The sialylated glycans were further purifiedby adsorbing them to microcrystalline cellulose inn-butanol:ethanol:water (10:1:2, v/v), washing with the same solvent,and eluting by 50% ethanol:water (v/v). All the above steps wereperformed on miniaturized chromatography columns and small elution andhandling volumes were used. The glycan analysis method was validated bysubjecting human cell samples to analysis by five different persons. Theresults were highly comparable, especially by the terms of detection ofindividual glycan signals and their relative signal intensities, showingthat the reliability of the present methods is suitable for comparinganalysis results from different cell types.

Mass spectrometry and data analysis. MALDI-TOF mass spectrometry wasperformed with a Bruker Ultraflex TOF/TOF instrument (Bruker, Germany)essentially as described (Saarinen et al., 1999). Relative molarabundancies of both neutral and sialylated glycan components can beaccurately assigned based on their relative signal intensities in themass spectra (Naven and I-Harvey, 1996; Papac et al., 1996; Saarinen etal., 1999; Harvey, 1993). Each step of the mass spectrometric analysismethods were controlled for their reproducibility by mixtures ofsynthetic glycans or glycan mixtures extracted from human cells. Themass spectrometric raw data was transformed into the present glycanprofiles by carefully removing the effect of isotopic patternoverlapping, multiple alkali metal adduct signals, products ofelimination of water from the reducing oligosaccharides, and otherinterfering mass spectrometric signals not arising from the originalglycans in the sample. The resulting glycan signals in the presentedglycan profiles were normalized to 100% to allow comparison betweensamples. Quantitative difference between two glycan profiles (%) wascalculated according to the equation:

$\begin{matrix}{{{difference} = {\frac{1}{2}{\sum\limits_{i = 1}^{n}{{p_{i,a} - p_{i,b}}}}}},} & (2)\end{matrix}$

wherein p is the relative abundance (%) of glycan signal i in profile aor b, and n is the total number of glycan signals.

Glycosidase analysis. The neutral N-glycan fraction was subjected todigestion with Jack bean α-mannosidase (Canavalia ensiformis; Sigma,USA) essentially as described (Saarinen et al., 1999). The specificityof the enzyme was controlled with glycans isolated from human tissues aswell as purified oligosaccharides.

NMR methods. For NMR analysis, larger amounts of hESC were grown onmouse feeder cell (MEF) layers. The purity of the collected hESC sample(about 70%), was lower than in the mass spectrometry samples grown onHFF. However, the same H₅₋₉N₂ glycans were the major neutral N-glycansignals in both MEF and hESC. The isolated glycans were further purifiedfor the analysis by gel filtration high-pressure liquid chromatographyin a column of Superdex peptide HR 10/30 (Amersham), with water (neutralglycans) or 50 mM NH₄HCO₃ (sialylated glycans) as the eluant at a flowrate of 1 ml/min. The eluant was monitored at 214 nm, andoligosaccharides were quantified against external standards. The amountof N-glycans in NMR analysis was below five nanomoles.

Statistical procedures. Glycan score distributions of all threedifferentiation stages (hESC, EB, and st.3) were analyzed by theKruskal-Wallis test. Parise comparisons were performed by the 2-tailedStudent's t-test with Welch's approximation and 2-tailed Mann-Whitney Utest. A p value less than 0.05 was considered significant.

Lectin staining. Fluorescein-labeled lectins were from EY Laboratories(USA) and the stainings were performed essentially after manufacturer'sinstructions. The specificity of the staining was controlled in parallelexperiments by inhibiting lectin binding with specific oligo- andmonosaccharides.

Results

Mass Spectrometric Profiling of the hESC N-glycome

In order to generate glycan profiles of hESC, embryonic bodies, andfurther differentiated cells, a MALDI-TOF mass spectrometry basedanalysis was performed as outlined in FIG. 32. We focused on the mostcommon type of protein post-translational modifications, theasparagine-linked glycans (N-glycans), which were enzymatically releasedfrom cellular glycoproteins. During glycan isolation and purification,the total N-glycan pool was separated by an ion-exchange step intoneutral N-glycans and sialylated N-glycans. These two glycan fractionswere then analyzed separately by mass spectrometric profiling (FIG. 33),which yielded a global view of the N-glycan repertoire of the samples.The relative abundances of the observed glycan signals were determinedbased on their relative signal intensities (Naven and Harvey, 1996;Papac et al., 1996; Saarinen et al., 1999), which allowed quantitativecomparison of glycome differences between samples. Over one hundredN-glycan signals were detected from each cell type.

The proposed monosaccharide compositions corresponding to the detectedmasses of each individual signal in FIG. 33 is indicated by letter code.However, it is important to realize that many of the mass spectrometricsignals in the present analyses include multiple isomeric structures andthe 100 most abundant signals very likely represent hundreds ofdifferent molecules. For example, the common hexoses (H) occurring inhuman N-glycans include D-mannose, D-galactose, and D-glucose (which allhave a residue mass of 162.05 Da), and common N-acetylhexosamines (N)include both N-acetyl-D-glucosamine and N-acetyl-D-galactosamine (203.08Da); deoxyhexoses (F) are typically L-fucose residues (146.06 Da).

In most of the previous glycomic studies of other mammalian tissues theisolated glycans have been derivatized (permethylated) prior to massspectrometric profiling (Sutton-Smith et al., 2002; Dell and Morris,2001; Consortium for Functional Glycomics,http://www.functionalglycomics.org) or chromatographic separation(Callewaert et al., 2004). However, in the present study we chose todirectly analyze picomolar quantities of unmodified glycans andincreased sensitivity was attained by omitting the derivatization andthe subsequent additional purification steps. Further, instead ofstudying the glycan signals one at a time, we were able tosimultaneously study all the glycans present in the unmodified glycomesby nuclear magnetic resonance spectroscopy (NM) and specific glycosidaseenzymes. The present data demonstrate that mass spectrometric profilingcan be used in the quantitative analysis of total glycomes, especiallyto pin-point the major glycosylation differences between relatedsamples.

Overview of the hESC N-glycome: Neutral N-glycans

Neutral N-glycans comprised approximately two thirds of the combinedneutral and sialylated N-glycan pools. The 50 most abundant neutralN-glycan signals of the hESC lines are presented in FIG. 33 a (greycolumns). The similarity of the profiles, which is indicated by theminor variation in the glycan signals, suggest that the four cell linesclosely resemble each other. For example, 15 of the 20 most abundantglycan signals were the same in every hESC line. These 15 neutralN-glycan signals typical for the hESC N-glycome are listed in Table 38.The five most abundant signals comprised 76% of the neutral N-glycans ofhESC and dominated the profile.

Sialylated N-glycans

All N-glycan signals in the sialylated N-glycan fraction (FIG. 33 b,grey columns) contain sialic acid residues (S: N-acetyl-D-neuraminicacid, or G: N-glycolyl-D-neuraminic acid). The 50 most abundantsialylated N-glycans in the four hESC lines showed more variationbetween individual cell lines than the neutral N-glycans. However, thefour cell lines again resembled each other. The group of five mostabundant sialylated N-glycan signals was the same in every cell line:S₁H₅N₄F₁, S₁H₅N₄F₂, S₂H₅N₄F₁, S₁H₅N₄, and S₁H₆N₅F₁ (for abbreviationssee FIG. 33). The 15 sialylated N-glycan signals common to all the hESClines are listed in Table 39. The majority (61%, in eight signals) ofthe sialylated glycan signals contained the H₅N₄ core composition anddiffered only by variable amounts of sialic acid (S or G) anddeoxyhexose (F) residues. Similarly, another common core structure wasH₆N₅ (12%, in seven signals). This highlights the biosyntheticmechanisms leading to the total spectrum of N-glycan structures incells: N-glycans typically consist of common core structures that aremodified by the addition of variable epitopes (FIG. 35).

Importantly, we were able to detect N-glycans containingN-glycolylneurarminic acid (G), for example glycans G₁H₅N₄, G₁S₁H₅N₄,and G₂H₅N₄, in the hESC samples. N-glycolylneuraminic acid haspreviously been reported in hESC as an antigen transferred from culturemedia containing animal-derived materials (Martin et al., 2005).Accordingly, the serum replacement medium used in the presentexperiments contained bovine serum proteins.

Variation Between Individual Cell Lines

Although the four hESC lines shared the same overall N-glycan profile,there was cell line specific variation within the profiles. Individualglycan signals unique to each cell line were detected, indicating thatevery cell line was slightly different from each other with respect tothe approximately one hundred most abundant N-glycan structures theysynthesized.

In general, the 30 most common N-glycan signals in each hESC lineaccounted for circa 85% of the total detected N-glycans, and represent auseful approximation of the hESC N-glycome (Tables 38 and 39). In otherwords, more than five out of six glycoprotein molecules isolated fromany of the present hESC lines would carry such N-glycan structures.

Transformation of the N-glycome During hESC Differentiation

A major goal of the present study was to identify glycan structures thatwould be specific to either stem cells or differentiated cells, andcould therefore serve as differentiation stage markers. In order todetermine whether the hESC N-glycome undergoes changes duringdifferentiation, the N-glycan profiles obtained from hESC, EB, and stage3 differentiated cells were compared (FIG. 33). The profiles of thedifferentiated cell types (EB and st.3) were significantly differentfrom the profiles of undifferentiated hESC, indicated by non-overlappingdistribution bars in many glycan signals. Further, there were manysignals present in both hESC and EB that were not detected in stage 3differentiated cells. Overall, 10% of the glycan signals present in hESChad disappeared in stage 3 differentiated cells. Simultaneously numerousnew signals appeared in EB and stage 3 differentiated cells. Theirproportion in EB and stage 3 differentiated cells was 14% and 16%,respectively. The glycan signals that were characteristic for hESC weretypically decreased in the EB and had further decreased or totallydisappeared in stage 3 differentiated cells. However, among the mostcommon one hundred glycan signals there were no hESC signals that wouldnot have been expressed in EB, suggesting that the EB N-glycome is anintermediate between hESC and stage 3 differentiated cells.

Taken together, differentiation induced the appearance of new N-glycantypes while earlier glycan types disappeared. Further, we found that themajor hESC-specific N-glycosylation features were not expressed asdiscrete glycan signals, but instead as glycan signal groups that werecharacterized by a specific monosaccharide composition feature (seebelow). In other words, differentiation of hESC into EB induced thedisappearance of not only one but multiple glycan signals withhESC-associated features, and simultaneously also the appearance ofglycan signal groups with other features associated with thedifferentiated cell types.

The N-glycan profiles of the differentiated cells were alsoquantitatively different from the undifferentiated hESC profiles. Apractical way of quantifying the differences between individual glycanprofiles is to calculate the sum of the signal intensity differencesbetween two cell profiles (see Methods). According to this method, theEB neutral and sialylated N-glycan profiles had undergone a quantitativechange of 14% and 29% from the hESC profiles, respectively. Similarly,the stage 3 differentiated cell neutral and sialylated N-glycan profileshad changed by 15% and 43% from the hESC profiles, respectively. Thisindicates that upon differentiation of hESC into stage 3 differentiatedcells, nearly half of the total sialylated N-glycans present in thecells were transformed into different molecular structures, whilesignificantly smaller proportion of the neutral N-glycan molecules werechanged during the differentiation process. Taking into account that theproportion of sialylated to neutral N-glycans in hESC was approximately1:2, the total N-glycome change was approximately 25% during thetransition from hESC to stage 3 differentiated cells. Again, theN-glycan profile of EB appeared to lie between hESC and stage 3differentiated cells.

The data indicated that the hESC N-glycome consisted of two discreteparts regarding propensity to change during hESC differentiation—aconstant part of circa 75% and a changing part of circa 25%. In order tocharacterize the associated N-glycan structures, and to identify thepotential biological roles of the constant and changing parts of theN-glycome, we performed structural analyses of the isolated hESCN-glycan samples.

Structural Analyses of the Major hESC N-glycans: Preliminary StructureAssignment Based on Monosaccharide Compositions

Human N-glycans can be divided into the major biosynthetic groups ofhigh-mannose type, hybrid-type, and complex-type N-glycans. To determinethe presence of these N-glycan groups in hESC and their progeny,assignment of probable structures matching the monosaccharidecompositions of each individual signal was performed utilizing theestablished pathways of human N-glycan biosynthesis (Kornfeld andKornfeld, 1985; Schachter, 1991). Here, the detected N-glycan signalswere classified into four N-glycan groups according to the number of Nand H residues in the proposed compositions as shown in FIG. 35 a: 1)high-mannose type and 2) low-mannose type N-glycans, which are bothcharacterized by two N residues (N=2), 3) hybrid-type or monoantennaryN-glycans, which are classified by three N residues (N=3), and 4)complex-type N-glycans, which are characterized by four or more Nresidues (N≧4) in their proposed monosaccharide compositions. This is anapproximation: for example, in addition to complex-type N-glycans alsohybrid-type and monoantennary N-glycans may contain more than three Nresidues.

The data was analyzed quantitatively by calculating the percentage ofglycan signals in the total N-glycome belonging to each structure group(Table 41, rows A-E and J-L; FIG. 35 b). The quantitative changes in thestructural groups reflect the relative activities of differentbiosynthetic pathways in each cell type. For example, the proportion ofhybrid-type or monoantennary N-glycans was increased when hESCdifferentiated into EB. In general, the relative proportions of mostglycan structure classes remained approximately constant through thehESC differentiation process, which indicated that both hESC and thedifferentiated cell types were capable of equally sophisticatedN-glycosylation. The high proportion of N-glycans classified aslow-mannose N-glycans in all the studied cell types was somewhatsurprising in the light of earlier published studies of humanN-glycosylation. However, previous studies had not explored the totalN-glycan profiles of living cells. We have detected significant amountsof low-mannose N-glycans also in other human cells and tissues, and theyare not specific to hESC (T.S., A.H., M.B., A.O., J.H., J.N, J. S. etal., unpublished results).

Verification of Structure Assignments by Enzymatic Degradation andNuclear Magnetic Resonance Spectroscopy

In order to verify the validity of the glycan structure assignments madebased on the detected mass and the probable monosaccharide compositionswe performed enzymatic degradation and proton nuclear magnetic resonancespectroscopic analyses (¹H-NMR) of selected neutral and sialylatedN-glycans.

For the validation of neutral N-glycans we chose glycans with 5-9 hexose(H) and two N-acetylhexosamine (N) residues in their monosaccharidecompositions (H₅N₂, H₆N₂, H₇N₂, H₈N₂, and H₉N₂) which were the mostabundant N-glycans in all studied cell types (FIG. 33 a). Themonosaccharide compositions suggested (FIG. 35 a) that these glycanswere high-mannose type N-glycans (Kornfeld and Kornfeld, 1985). To testthis hypothesis, neutral N-glycans from stem cell and differentiatedcell samples were treated with α-mannosidase, and analyzed both beforeand after the enzymatic treatment (data not shown). The glycans inquestion were degraded and the corresponding signals disappeared fromthe mass spectra, indicating that they contained a-linked mannoseresidues.

The neutral N-glycan fraction was further analyzed by nanoscale protonnuclear magnetic resonance spectroscopic analysis (¹H-NMR). In theobtained ¹H-NMR spectrum of the hESC neutral N-glycans signalsconsistent with high-mannose type N-glycans were detected, supportingthe conclusion that they were the major glycan components in the sample.

Both α-mannosidase and NMR experiments indicated that the H₅₋₉N₂ glycansignals corresponded to high-mannose type N-glycans. From the data inFIG. 33 a it could be estimated that they constituted half of all thedetected glycoprotein N-glycans in hESC. This is in accordance with theestablished role of high-mannose type N-glycans in human cells (Heleniusand Aebi, 2001, 2004). The presence of such constitutively expressedN-glycans also explained why the neutral N-glycan profiles did notchange to the same extent as the sialylated N-glycan profiles duringdifferentiation.

For the validation of structure assignments among the sialylatedN-glycans we noted that the majority of the sialylated N-glycan signalsisolated from hESC were characterized by the N≧4 monosaccharidecomposition (FIG. 33 a), which suggested that they were complex-typeN-glycans (FIG. 35). In the ¹H-NMR analysis N-glycan backbone signalsconsistent with biantennary complex-type N-glycans were the majordetected signals, in line with the assigment made based on theexperimental monosaccharide compositions. The present results indicatedthat the classification of the glycan signals within the total N-glycomedata could be used to construct an approximation of the whole N-glycome.However, such classification should not be applied to the analysis ofsingle N-glycan signals.

Differentiation Stage Associated Structural Glycosylation Features

The glycan signal classification described above indicated changes inthe core sequences of N-glycans. The present data also suggested thatthere were differences in variable epitopes added to the N-glycan corestructures i.e. glycan features present in many individual glycansignals. In order to quantify such glycan structural features, theN-glycome data were further classified into glycan signal groups thatshare similar features in their proposed monosaccharide compositions(Table 41, rows F-I and M-P). As a result, the majority of thedifferentiation-associated glycan signals in the EB and stage 3differentiated cell samples fell into different groups than the hESCspecific glycans. Glycan signals with complex fucosylation (Table 41,row N) were associated with undifferentiated hESC, whereas glycansignals with potential terminal N-acetylhexosamine (Table 41, rows H andP) were associated with the differentiated cells.

Complex Fucosylation of N-glycans is Characteristic of hESC

Differentiation stage associated changes in the sialylated N-glycanprofile were more drastic than in the neutral N-glycan fraction and thegroup of five most abundant sialylated N-glycan signals was different atevery differentiation stage (FIG. 33 b). In particular, there was asignificant differentiation-associated decrease in the relative amountsof glycans S₁H₅N₄F₂ and S₁H₅N₄F₃ as well as other glycan signals thatcontained at least two deoxyhexose residues (F≧2) in their proposedmonosaccharide compositions. In contrast, glycan signals such as S₂H₅N₄that contained no F were increased in the differentiated cell types. Theresults suggested that sialylated N-glycans in undifferentiated hESCwere subject to more complex fucosylation than in the differentiatedcell types (Table 41, row N).

The most common fucosylation type in human N-glycans isα1,6-fucosylation of the N-glycan core structure. The NMR analysis ofthe sialylated N-glycan fraction of hESC also revealed α1,6-fucosylationof the N-glycan core as the most abundant type of fucosylation. In theN-glycans containing more than one fucose residue, there must have beenother fucose linkages in addition to the α1,6-linkage (Staudacher etal., 1999). The F≧2 structural feature decreased as the cellsdifferentiated, indicating that complex fucosylation was characteristicof undifferentiated hESC.

N-glycans with Terminal N-acetylhexosamine Residues Become More Commonwith Differentiation

A group of N-glycan signals which increased during differentiationcontained equal amounts of N-acetylhexosamine and hexose residues (N═H)in their monosaccharide composition, e.g. S₁H₅N₅F₁. This was consistentwith structures containing non-reducing terminal N-acetylhexosamineresidues. Usually N-glycan core structures contain more hexose thanN-acetylhexosamine residues. However, if complex-type N-glycans containterminal N-acetylhexosamine residues that are not capped by hexoses,their monosaccharide compositions change to either the N═H or the N>H(FIG. 35 a). EB and stage 3 differentiated cells showed increasedamounts of potential terminal N-acetylhexosamine structures, of whichthe N═H structural feature was increased in both neutral and sialylatedN-glycan pools (Table 41, rows I and P), whereas the N>H structuralfeature was elevated in the neutral N-glycan pool, but decreased in thesialylated N-glycan pool during differentiation (Table 41, rows H andO).

Glycome Profiling can Identify the Differentiation Stage of hESC

The analysis of glycome profiles indicated that the studied hESC linesand differentiated cells had differentiation stage specific N-glycanfeatures. However, the data also demonstrated that N-glycan profiles ofthe individual hESC lines were different from each other and inparticular the hESC line FES 22 was different from the other three stemcell lines (Table 41, rows C and I). To test whether the obtainedN-glycan profiles could be used to generate an algorithm that woulddiscriminate between hESC and differentiated cells even taking intoaccount cell line specific variation, an analysis was performed usingthe data of Table 41. The hESC line FES 29 and embryoid bodies derivedfrom it (EB 29) were selected as the training group for the calculation.The algorithm glycan score (Equation 1) was defined as the sum of thosestructural features that were at least two times greater in FES 29 thanin EB 29 (row N in Table 41), from which the sum of the structuralfeature percentages that were at least two times greater in EB 29 thanin FES 29 was subtracted (rows C, I, J, and P in Table 41):

glycan score=N−(C+I+J+P),  (1)

wherein the letters refer to the row numbering of Table 41.

The algorithm was then applied to the other samples that served as thetest group in the analysis and the results are described graphically inFIG. 36. The differentiated cell samples (EB and stage 3) weresignificantly discriminated from hESC with p<0.01 (2-tailed Student'st-test with Welch's approximation, p=0.0018). The stage 3 differentiatedcell samples were also significantly separated from the EB samples withp<0.01 (2-tailed Marm-Whitney U test, p=0.0022). This suggested that thehESC N-glycan profiles were similar at the glycome level despite ofindividual differences at the level of distinct glycan signals. Theresult also suggested that glycome profiling is a potential tool formonitoring the differentiation status of stem cells.

The identified hESC Glycans can be Targeted at the Cell Surface

From a practical perspective stem cell research would be best served bythe identification of target structures on cell surface. To investigatewhether individual glycan structures we had identified would beaccessible to reagents targeting them at the cell surface we performedlectin labelling of two candidate structure types. Lectins are proteinsthat recognize glycans with specificity to certain glycan structuresalso in hESC (Venable et al., 2005). To study the localization of glycancomponents in hESC, stem cell colonies grown on mouse feeder cell layerswere labeled in vitro by fluorescein-labelled lectins (FIG. 37). ThehESC cell surfaces were clearly labeled by Maackia amurensis agglutinin(MAA) that recognizes structures containing α2,3-linked sialylation,indicating that sialylated glycans are abundant on the hESC cell surface(FIG. 37 a). Such glycans would thus be available for recognition bymore specific glycan-recognizing reagents such as antibodies. Incontrast, the cell surfaces were not labelled by Pisum sativumagglutinin (PSA) that recognizes α-mannosylated glycans (FIG. 37 b).However, PSA labelled the cells after permeabilization (data not shown),suggesting that the mannosylated N-glycans in hESC were localized inintracellular cell compartments such as the endoplasmic reticulum (ER)or the Golgi complex (FIG. 37 c). Interestingly, the mouse fibroblastcells showed complementary staining patterns, suggesting that theselectin reagents efficiently discriminated between hESC and feeder cells.Together the results suggested that the glycan structures we identifiedcould be utilized to design specific reagents targeting hESC.

Comparative Analysis of the N-glycome

Although the N-glycan profiles of the four hESC lines share a similaroverall profile shape, there was cell line specific variation in theN-glycan profiles. Individual glycan signals unique to each cell linewere found, indicating that every cell line was slightly different fromeach other with respect to the approximately one hundred most abundantglycan structures they synthesize. This is represented in FIG. 34 a asVenn diagrams combining all the detected glycan signals from both theneutral and the acidic N-glycan fractions. FES 29 and FES 30 werederived from sibling embryos, but their N-glycan profiles did notresemble each other more than they resembled FES 21 in the Venn diagram.Furthermore, FES 30 that has the karyotype XX did not differsignificantly from the three XY hESC lines.

In order to determine whether the hESC N-glycome undergoes changesduring differentiation, N-glycan profiles obtained from hESC, EB, andstage 3 differentiated cells were compared (FIG. 33). The N-glycanprofiles of the differentiated cell types (EB and st.3) differedsignificantly from the profiles of undifferentiated hESC, which isindicated by non-overlapping distribution bars in many glycan signals.There were many signals in common between hESC and EB that disappearedin stage 3 differentiated cells, as described in the Venn diagram (FIG.34 b). Overall, 17% of the glycan signals present in hESC disappeared inEB, and in stage 3 differentiated cells 58% of the original N-glycansignals disappeared. Simultaneously numerous new signals appeared in EBand stage 3 differentiated cells. Their proportion in EB and stage 3differentiated cells was 24% and 10%, respectively. This indicates thatdifferentiation induced the appearance of new N-glycan types whileearlier glycan types disappeared. The 19 N-glycan signals specific tothe hESC samples are listed in Table 40.

Discussion

In the present study, novel mass spectrometric methods were applied tothe first structural analysis of human embryonic stem cell N-glycanprofiles. Previously, such investigation of whole cell glycosylation hasnot been feasible due to the lack of methods with sufficiently highsensitivity to analyze the scarce stem cells. The present method wasvalidated for samples of approximately 100 000 cells and the glycanprofiles of the analyzed cell types were consistent throughout multiplesamples. The objective in the use of the present method was to provide aglobal view on the glycome profile, or a “fingerprint” of hESCglycosylation, rather than to present the stem cell glycome in terms ofthe molecular structures of each glycan component. However, changesobserved in the N-glycan profiles provide vast amount of informationregarding hESC glycosylation and its changes during differentiation, andallows rational design of detailed structural studies of selected glycancomponents or glycan groups.

The results indicate that a defined group of N-glycan signals dominatethe hESC N-glycome and form a unique stem cell glycan profile. It seemsthat specific monosaccharide compositions were favored over the possiblealternatives by the hESC N-glycan biosynthetic machinery. For example,the fifteen most abundant neutral N-glycan signals and fifteen mostabundant sialylated N-glycan signals in hESC together comprised over 85%of the N-glycome. Further, different glycan structures were favoredduring the differentiation of the cells. This suggests that N-glycanbiosynthesis in hESC is a controlled and predetermined process. Ashundreds of genes, consisting of up to 1% of the human genome, areinvolved in glycan biosynthesis (Haltiwanger and Lowe, 2004), a futurechallenge is to characterize the regulatory processes that control hESCglycosylation during differentiation into specialized cell types.

Based on our results the hESC N-glycome seems to contain both a constantpart consisting of “housekeeping glycans”, and a changeable part thatwas altered when the hESC differentiated (FIG. 33). The constant partseemed to contain mostly high-mannose type and biantennary complex-typeN-glycans. Such “housekeeping” glycans may need to be present at alltimes for the maintenance of basic cellular processes. Significantly,25% (50% if high-mannose glycans are excluded) of the total N-glycanprofile of hESC changed during their differentiation. This indicatesthat during differentiation hESC dramatically change both theirappearance towards their environment and possibly also their owncapability to sense and respond to exogenous signals.

Our data show that the differentiation-associated change in theN-glycome was generated by addition of variable epitopes on similarN-glycan core compositions. For example, the present lectin stainingexperiments demonstrated that sialylated glycans were abundant on thecell surface of hESC, indicating that they are potential targets fordevelopment of more specific recognition reagents. In contrast, theconstantly expressed mannosylated glycans were found to reside mainlyinside the cells. It seems plausible that knowledge of the changingsurface glycan epitopes could be utilized as a basis in developingreagents and culture systems that would allow improved identification,selection, manipulation, and culture of hESC and their progeny. We arecurrently characterizing the stem cell specific glycosylation changes atthe level of individual molecular structures.

The specific cellular glycan structures perform their functions mainlyby 1) acting as ligands for specific glycan receptors (Kilpatrick, 2002;Zanetta and Vergoten, 2003), 2) functioning as structural elements ofthe cell (Imperiali and O'Connor, 1999), and 3) modulating the activityof their carrier proteins and lipids (Varki, 1993;). More than half ofall proteins are glycosylated. Consequently, a global change inprotein-linked glycan biosynthesis can simultaneously modulate theproperties of multiple proteins. It is likely that the large changes inN-glycans during hESC differentiation have major influences on a numberof cellular signaling cascades and affect in profound fashion biologicalprocesses within the cells. Our data may provide insight into theregulation of some of these processes.

The major hESC specific glycosylation feature we identified was thepresence of more than one deoxyhexose residue in N-glycans, indicatingcomplex fucosylation. Fucosylation is known to be important in celladhesion and signalling events (Becker and Lowe, 2003) as well asessential for embryonic development. Knock-out of the N-glycan coreα1,6-fucosyltransferase gene FUT8 leads to postnatal lethality in mice(Wang et al., 2005), and mice completely deficient in fucosylated glycanbiosynthesis do not survive past early embryonic development (Smith etal., 2002). Fucosylation defects in humans cause a disease known asleukocyte adhesion deficiency (LAD; Luhn et al., 2001).

Fucosylated glycans such as the SSEA-1 antigen have previously beenassociated with both mouse embryonic stem cells (mESC) and humanembryonic carcinoma cells (EC; Muramatsu and Muramatsu, 2004), but notwith hESC. In addition, structurally related Le^(x) oligosaccharides areable to inhibit embryonic compaction (Fenderson et al., 1984),suggesting that fucosylated glycans are directly involved incell-to-cell contacts during embryonic development. Theα1,3-fucosyltransferase genes indicated in the synthesis of theembryonic Le^(x) and SSEA-1 antigens are FUT4 and FUT9 (Nakayama et al.,2001; Kudo et al., 2004). Interestingly, the published gene expressionprofiles for the same hESC lines as studied here (Skottman et al., 2005)have demonstrated that three human fucosyltransferase genes, FUT1, FUT4,and FUT8 are expressed in hESC, and that FUT1 and FUT4 are overexpressedin hESC when compared to EB. The known specificities of thesefucosyltransferases (Mollicone et al., 1995) correlate with our findingsof simple fucosylation in EB and complex fucosylation in hESC (FIG. 38).Taken together, although hESC do not express the specific glycolipidantigen recognized by the SSEA-1 antibody, they share with mESC thecharacteristic feature of complex fucosylation and may have conservedthe biological functions of fucosylated glycan epitopes.

New N-glycan forms emerged in EB and stage 3 differentiated cells. Thesestructural features included additional N-acetylhexosamine residues,potentially leading to new N-glycan terminal epitopes. Anotherdifferentiation-associated feature was an increase in the molarproportions of hybrid-type or monoantennary N-glycans. Biosynthesis ofhybrid-type and complex-type N-glycans has been demonstrated to bebiologically significant for embryonic and postnatal development in themouse (Ioffe and Stanley, 1994 PNAS; Metzler et al., 1994 EMBO J; Wanget al., 2001 Glycobiology; Akama et al., 2006 PNAS). The preferentialexpression of complex-type N-glycans in hESC and then the change in thedifferentiating EB to express more hybrid-type or monoantennaryN-glycans may thus be significant for the process of stem celldifferentiation.

Human embryonic stem cell lines have previously been demonstrated tohave a common genetic stem cell signature that can be identified usinggene expression profiling techniques (Skottman et al., 2005; Sato etal., 2003; Abeyta et al., 2004; Bhattacharya et al., 2004). Suchsignatures have been proposed to be utilized in the characterization ofcell lines. The present report provides the first glycomic signaturesfor hESC. The profile of the expressed N-glycans might be a useful toolfor analyzing and classifying the differentiation stage in associationwith gene and protein expression analyses. Here we demonstrate that theglycan score algorithm was able to reliably differentiate cell samplesof separate differentiation stage (FIG. 37). Glycome profiling may be amore sensitive measure of the cell status than any single cell surfacemarker. Such a method might be especially useful for the quality controlof hESC-based cell products. However, further analysis of the hESCglycome may also lead to discovery of novel glycan antigens that couldbe used as stem cell markers in addition to the commonly used SSEA andTra glycan antigens.

In conclusion, hESC have a unique glycome which undergoes major changeswhen the cells differentiate. Information regarding the specific glycomemay be utilized in developing reagents for the targeting of these cellsand their progeny. Future studies investigating the developmental andmolecular regulatory processes resulting in the observed glycan profilesmay provide significant insight into mechanisms of human development andregulation of glycosylation.

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Example 24 Gene Expression and Glycome Profiling of Human Embryonic StemCells Results and Discussion

Obtaining of the gene expression data from the hESC lines FES 21, 22,29, and 30 has been described (Skottnan et al., 2005) and the presentdata was produced essentially similarity. The results of the geneexpression profiling analysis with regard to a selection of potentiallyglycan-processing and accessory enzymes are presented in Table 42, wheregene expression is both qualitatively determined as being present (P) orabsent (A) and quantitatively measured in comparison to embryoid bodies(EB) derived from the same cell lines.

Fucosyltransferase expression levels. Three fucosyltransferasetranscripts were detected in hESC: FUT1 (α1,2-fucosyltmmsferase;increased in all FES cell lines), FUT4 (α1,3-fucosyltransferase IV;increased in all FES cell lines), and FUT8 (N-glycan coreα1,6-fucosyltransferase).

Hexosaminyltransferase expression levels. The following transcripts inthe selection of Table 42 were detected in hESC: MGAT3, MGAT2 (increasedin three FES cell lines), MGAT1, GNT4b, β3GlcNAc-T5, β3GlcNAc-T7,β3GlcNAc-T4 (present in two FES cell lines), β6GlcNAcT (increased in oneFES cell line), iβ3GlcNAcT, globosideT, and α4GlcNAcT (present in twoFES cell lines).

Other gene epression levels. The following transcripts in the selectionof Table 42 were detected in hESC: AER1 (increased in all FES celllines), AGO61, β3GALT3, MAN1C1, and LGALS3.

Example 25 Analysis of Human and Murine Fibroblast Feeder Cells

Murine (mEF) and human (hEF) fibroblast feeder cells were prepared andtheir N-glycan fractions analyzed as described in the precedingExamples.

Results and Discussion

FIG. 43 shows the major neutral N-glycan fraction glycan signals of hEFand mEF. FIG. 44 shows the glycan grouping of neutral N-glycan fractionglycan signals of hEF and mEF. FIG. 45 shows the glycan grouping ofacidic N-glycan fraction glycan signals of hEF and mEF. The mEF and hEFcells differed significantly from each other in their glycan profiles.

The results showed that mEF and hEF cellular N-glycan fractions differsignificantly from each other. The differencies include differentialproportions of glycan groups, major glycan signals, and the glycanprofiles obtained from the cell samples. In addition, the majordifference is the presence of Galα3Gal epitopes in the mEF cells, asdiscussed in the preceding Examples of the present invention.

Example 26 The glycome of Human Embryonic Stem Cells Reflects TheirDifferentiation Stage Summary

Complex carbohydrate structures, glycans, are elementary components ofglycoproteins, glycolipids, and proteoglycans. These glycoconjugatesform a layer of glycans that covers all human cell surfaces and formsthe first line of contact towards the cell's environment. Glycanstructures called stage specific embryonic antigens (SSEA) are used toassess the undifferentiated stage of embryonic stem cells. However, thewhole spectrum of stem cell glycan structures has remained unknown,largely due to lack of suitable analysis technology. We describe thefirst global study of glycoprotein glycans of human embryonic stemcells, embryoid bodies, and further differentiated cells by MALDI-TOFmass spectrometric profiling. The analysis reveals how certainasparagine-linked glycan structures characteristic to stem cells arelost during differentiation while new structures emerge in thedifferentiated cells. The results indicate that human embryonic stemcells have a unique glycome and that their differentiation stage can beidentified by glycome analysis. We suggest that knowledge about stemcell specific glycan structures can be used for e.g. purification,manipulation, and quality control of stem cells.

Materials & Methods

Human embryonic stem cell lines. Four Finnish hESC lines, FES 21, FES22, FES 29, and FES 30 (Skottman et al., 2005. Stem cells 23:1343-56)were used in the present study. These lines are included in theInternational Stem Cell Initiative (Andrews et al., 2005. Nat.Biotechnol. 23:795-7). The cells were propagated on human foreskinfibroblast (hFF) feeder cells in serum-free medium (Knockout™,Gibco/Invitrogen). In FACS analyses 70-90% of cells from mechanicallyisolated colonies were typically Tra 1-60 and Tra 1-81 positive (notshown). Cells differentiated into embryoid bodies (EB, stage 2differentiated) and further differentiated cells grown out of the EB asmonolayers (stage 3 differentiated) were used for comparison againsthESC. The differentiation protocol favors the development ofneuroepithelial cells while not directing the differentiation intodistinct terminally differentiated cell types (Okabe et al., 1996. Mech.Dev. 59:89-102). EB derived from FES 30 had less differentiated celltypes than the other three EB. Stage 3 cultures consisted of aheterogenous population of cells dominated by fibroblastoid and neuronalmorphologies. For the glycome studies the cells were collectedmechanically, washed, and stored frozen until analysis.

In a preferred embodiment the invention is directed to the use of dataobtained embryoid bodies or ESC-cell line cultivated under conditionsfavouring neuroepithelial cells for search of specific structuresindicating neuroepithelial development, preferably by comparing thematerial with cell materials comprising neuronal and/or epithelial typecells. Asparagine-linked glycome profiling. Total asparagine-linkedglycan (N-glycan) pool was enzymatically isolated from about 100 000cells. The total N-glycan pool (picomole quantities) was purified withmicroscale solid-phase extraction and divided into neutral andsialylated N-glycan fractions. The N-glycan fractions were analyzed byMALDI-TOF mass spectrometry either in positive ion mode for neutralN-glycans or in negative ion mode for sialylated glycans (Saarinen etal., 1999, Eur. J. Biochem. 259, 829-840). Over one hundred N-glycansignals were detected from each cell type revealing the surprisingcomplexity of hESC glycosylation. The relative abundances of theobserved glycan signals were determined based on relative signalintensities (Harvey, 1993. Rapid Commun. Mass Spectrom. 7:614-9; Papacet al., 1996. Anal. Chem. 68:3215-23).

Results

In the present study, we analyzed the N-glycome profiles of hESC, EB,and st.3 differentiated cells (FIG. 39).

The similarity of the N-glycan profiles within the group of four hESClines suggested that the obtained N-glycan profiles are a description ofthe characteristic N-glycome of hESC. Overall, 10% of the 100 mostabundant N-glycan signals present in hESC disappeared in st.3differentiated cells, and 16% of the most abundant signals in st.3differentiated cells were not present in hESC. This indicates thatdifferentiation induced the appearance of new N-glycan types whileearlier glycan types disappeared. In quantitative terms, the differencesbetween the glycan profiles of hESC, EB, and st.3 differentiated cellswere: hESC vs. EB 19%, hESC vs. st.3 24%, and EB vs. st.3 12%.

The glycome profile data was used to design glycan-specific labelingreagents for hESC. The most interesting glycan types were chosen tostudy their expression profiles by lectin histochemistry as exemplifiedin FIG. 40 for the lectins that recognize either α2,3-sialylated(MAA-lectin, FIG. 40A.) binding to the hESC cells or α-mannosylatedglycans (PSA-lectin, FIG. 40B.) binding to the surfaces of feeder cells(MEF). The binding of the lectin reagents was inhibited by specificcarbohydrate inhibitors, sialylα2-lactose and mannose, respectively(FIG. 40C. and 40D.). The results are summarized in Table 49.

Table 49 further represent differential recognition feeder and stemcells by two other lectins, Ricinus communis agglutinin (RCA, ricinlectin), known to recognize especially terminal Galβ-structures,especially GalβAGlc(NAc)-type structures and peanut agglutinin (PNA)reconnizing Gal/GalNAc structures. The cell surface expression of ligandfor two other lectin RCA and PNA on hESC cells, but only RCA ligands offeeder cells.

The present results indicate and the invention is directed to the hESCglycans are potential targets for recognition by stem cell specificreagents. The invention is further directed to methods of specificrecognition and/or separation of hESC and differentiated cells such asfeeder cells by glycan structure specific reagents such as lectins.Human embryonic stem cells have a unique glycome that reflects theirdifferentiation stage. The invention is specifically directed toanalysis of cells according to the invention with regard todifferentiation stage.

The results were also used to generate an algorithm for identificationof hESC differentiation stage (FIG. 36). To test whether the obtainedN-glycan profiles could be used for reliable identification of hESC anddifferentiated cells even with the presence of sanple-to-samplevariation, a discrimination analysis was performed on the data The hESCline FES 29 and embryoid bodies derived from it (EB 29) were selected asthe training group for the calculation that effectively discriminatedthe two samples (FIG. 36):

glycan score=a−b−c,

wherein a is the sum of the relative abundances (%) of all signals withproposed compositions with two or more dHex (F≧2) in the sialylatedN-glycan fraction, b is the sum of the relative abundances (%) of allsignals with hybrid-type structures (ST=H), and c is the sum of therelative abundances (%) of all signals with proposed compositions withfive or more HexNAc and equal amounts of Hex and HexNAc (H=N≧5); seeTable 48 for structure codes and FIG. 39 for the dataset.

The resulting equation was applied to the other samples that served asthe test group in the analysis and the results are described graphicallyin FIG. 36. hESC and the differentiated cell samples were clearlydiscriminated from each other (p<0.01, Student's t test). Furthermore,the st.3 differentiated cell samples were separated from the EB samples(p<0.05, Mann-Whitney test). The predicted 95% confidence intervals(assuming normal distribution of glycan scores within each cell type)are shown for the three cell types, indicating that a calculated glycanscore has potential to discriminate all three cell types. At 96%confidence interval, hESC and the differentiated cell types (EB andst.3) were still discriminated from each other (not shown in thefigure). The results indicate that glycome profiling is a tool formonitoring the differentiation status of stem cells.

Conclusions

The present data represent the glycome profiling of hESC:

-   -   hESC have a unique N-glycome comprising of over 100 glycan        components    -   Differentiation induces a major change in the N-glycome and the        cell surface molecular landscape of hESC

Utility of hESC glycome data:

-   -   Identification of new stem cell markers for e.g. antibody        development    -   Quality control of stem cell products    -   Identification of hESC differentiation stage    -   Control of variation between hESC lines    -   Effect of external factors and culture conditions on hESC status

Especially Preferred Uses of the Data are

Use of the hESC glycome for identification of specific cell surfacemarkers characteristic for the pluripotent hESCs.

The invention is directed to further analysis and production of presentand analogous glycome data and use of the methods for furtherindentification of novel stem cell specific glycosylation features andform the basis for studies of hESC glycobiology and its eventualapplications according to the invention

Example 27 Identification of Specific Glycosylation Signatures fromGlycan Profiles in Various Steps of Human Embryonic Stem CellDifferentiation

To identify differentiation stage specific N-glycan signals insialylated N-glycan profiles of hESC, EB, and stage 3 differentiatedcells (see Example 26 above), major signals specific to either theundifferentiated (FIG. 41) or differentiated cells (FIG. 42) wereselected based on their relative abundances in the database of the fourhESC lines, and the four EB and st.3 cell samples derived from the fourhESC lines, respectively. The selected glycan signal groups, from whereindifferent glycan signals have been removed, have reduced noise orbackground and less observation points, but have the resolving power.Such selected signal groups and their patterns in different sample typesserve as a signature for the identification of for example 1)undifferentiated hESC (FIG. 41), 2) differentiated cells, preferentiallytheir differentiation stage relative to hESC (FIG. 42), 3)differentiation lineage, such as the neuroectodermally enriched st3cells compared to the mixed cell population of EB (e.g. 1799), 4) glycansignals that are specific to hESC (e.g. 2953), 5) glycan signals thatare specific to differentiated cells (e.g. 2644), or 6) glycan signalsthat have individual i.e. cell line specific variation (e.g. 1946 incell line FES 22, 2133 in cell line FES 29, and 2222 in cell line FES30). Moreover, glycan signals can be identified that do not changeduring hESC differentiation, including major glycans that can beconsidered as housekeeping glycans in hESC and their progeny (e.g. 1257,1419, 1581, 1743, 1905 in FIG. 39A, and 2076 in FIG. 39.B). Proposedglycan compositions and structure groups for the signals are presentedin Table 48.

To further analyze the data and to find the major glycan signalsassociated in given hESC differentiation stage, two variables werecalculated for the comparison of glycan signals in the N-glycan profiledataset described above, between two samples:

1. absolute difference A=(S2−S1), and2. relative difference R=A/S1,wherein S1 and S2 are relative abundances of a given glycan signal insamples 1 (the four EB samples) and 2 (the four st.3 cell samples),respectively.

When A and R were calculated for the glycan profile datasets of the twocell types, and the glycan signals thereafter sorted according to thevalues of A and R, the most significant differing glycan signals betweenthe two samples could be identified. Among the fifty most abundantneutral N-glycan signals in the data (FIG. 39A), the following fivesignals experienced the highest relative change R in the transition fromEB to st3 differentiated cells in the dataset of four EB and four st.3cell samples: 1825 (R=5.8, corresponding to 6.8-fold increase), 1136(R=1.4, corresponding to 2.4 fold increase), 1339 (R=0.9, correspondingto 1.9 fold increase), 2142 (R=0.87, corresponding to 87% decrease), and2174 (R=0.56, corresponding to 56% decrease). Four of these signalscorresponded to complex-type structures (Table 48), indicating that themajor differing glycan structures were included in the complex-typeglycan group. However, the majority of the other complex-type glycansignals in the dataset were not observed to differ as significantlybetween the two cell types (i.e. they did not have large values of Aand/or R), indicating that the procedure was able to identify st.3 celland EB associated glycan subgroups within the whole complex-type glycangroup. The one signal corresponding to hybrid-type structures (1136) hadthe highest value of the absolute differences A among all the glycansignals in the neutral N-glycan profiles (A=0.48), indicating that alsothis signal had significance in the discrimination between the EB andst.3 cell samples in the studied dataset.

EB derived from the hESC line FES 30 were different in their overallN-glycan profiles compared to the other three EB samples (FIG. 39) andhad the differentiation-specific glycan score value closer to the hESCsamples (FIG. 36), correlating with the property of EB 30 having lessdifferentiated cell types than the other three EB. This was also seen indistinct glycan signals, e.g. 2222 in FIG. 39.B.

Example 28 Schematic Concepts of Glycome Change and Mass SpectrometricScreening Introduction to Glycomics

All human cell types have unique glycome—an entity of all glycans of thecell, present mainly on cell surface (FIG. 43) glycoproteins andglycolipids, including the SSEA and Tra glycan antigens. However, thewhole spectrum of hESC glycan structures (the stem cell glycome) isstill unknown. Glycans, the complex carbohydrate structures, are capableof great structural variation and their specific molecular structurescarry diverse biological information.

FIG. 43 represents schematically the changes of glycomes observed duringthe differentiation according to the invention. FIG. 32 representsschematically the glycome analysis, that was performed by MALDI-TOF massspectrometry of glycans released from cells.

Example 29 Influence of Lectins on Stem Cell Proliferation RateExperimental Procedures

Lectins (EY laboratories, USA) were passively adsorbed on 48-well plates(Nunclon surface, catalog No 150687, Nunc, Denmark) by overnightincubation in phosphate buffered saline.

Human bone marrow derived mesenchymal stem cells (BM MSC) were culturedin minimum essential α-medium (α-MEM) supplemented with 20 mM HEPES, 10%FCS, penicillin-streptomycin, and 2 mM L-glutamine (all from Gibco) on48-well plates coated with different lectins. Cells were cultivated inCell IQ (ChipMan Technologies, Tampere, Finland) at +37° C. with 5% CO₂.Images were taken every 15 minutes. Data were analyzed with Cell IQAnalyzer software by analyzer protocol built by Dr. Ulla Impola (FinnishRed Cross Blood Service, Helsinki, Finland).

Results and Discussion

The growth rates of BM MSC varied on different lectin-coated surfacescompared to each other and uncoated plastic surface (Table 50),indicating that proteins with different glycan binding specificitiesbinding to stem cell surface glycans specifically influence theirproliferation rate.

Lectins that had an enhancing effect on BM MSC growth rate included inorder of relative efficacy:

GS II (β-GlcNAc)>ECA (LacNAc/β-Gal)>PWA (I-branched poly-LacNAc)>LTA(α1,3-Fuc)>PSA (α-Man),wherein the preferred oligosaccharide specificities of the lectins areindicated in parenthesis. However, PSA was nearly equal to plastic inthe present experiments.

Lectins that had an inhibitory effect on BM MSC growth rate included inorder of relative efficacy:

RCA (β-Gal/LacNAc)>>UEA (α1,2-Fuc)>WFA (β-GalNAc)>STA (linearpoly-LacNAc)>NPA (α-Man)>SNA (α2,6-linked sialic acids)=MAA (α2,3-linkedsialic acids/α3′-sialyl LacNAc),wherein the preferred oligosaccharide specificities of the lectins areindicated in parenthesis. However, NPA, SNA, and MAA were nearly equalto plastic in the present experiments.

Example 30 Glycosphingolipid Glycans of Human Stem Cells ExperimentalProcedures

Samples from MSC, CB MNC, and hESC grown on mouse fibroblast feedercells were produced as described in the preceding Examples. Neutral andacidic glycosphingolipid fractions were isolated from cells essentiallyas described (Miller-Podraza et al., 2000). Glycans were detached byMacrobdella decora endoglycoceramidase digestion (Calbiochem, USA)essentially according to manuacturer's instructions, yielding the totalglycan oligosaccharide fractions from the samples. The oligosaccharideswere purified and analyzed by MALDI-TOF mass spectrometry as describedin the preceding Examples for the protein-linked oligosaccharidefractions. Proposed compositions for the oligosaccharides and signalnomenclature are presented in Tables 52 and 53 for the neutral andacidic glycan fractions, respectively.

Results and Discussion

Human Embryonic Stem Cells (hESC)

hESC neutral lipid glycans. The analyzed mass spectrometric profile ofthe hESC glycosphingolipid neutral glycan fraction is shown in FIG. 45.

Structural analysis of the major neutral lipid glycans. The six majorglycan signals, together comprising more than 90% of the total glycansignal intensity, corresponded to monosaccharide compositionsHex₃HexNAc₁ (730), Hex₃HexNAc₁dHex₁ (876), Hex₂HexNAc₁ (568),Hex₃HexNAc₂ (933), Hex₄HexNAc₁ (892), and Hex₄HexNAc₂ (1095).

In β1,4-galactosidase digestion, the relative signal intensities of 1095and 730 were reduced by about 30% and 10%, respectively. This suggeststhat 730 and 1095 contain minor components with non-reducing terminalβ1,4-Gal epitopes, preferably including the structures Galβ4GlcNAcLacand GalβGlcNAc[Hex₁HexNAc₁]Lac. The other major components were thusshown to contain other terminal epitopes. Further, the glycan signalHex₅HexNAc₃ (1460) was digested to Hex₃HexNAc₃ (1136), indicating thatthe original signal contained glycan structures containing two β1,4-Gal.

The major glycan signals were not sensitive to α-galactosidasedigestion.

In α1,3/4-fucosidase digestion, the signal intensity of 876 was reducedby about 10%, indicating that only a minor proportion of the glycansignal corresponded to glycans with α1,3- or α1,4-linked fucose residue.The major affected signal in the total profile was Hex₃HexNAc₁dHex₂(1022), indicating that it included glycans with either α1,3-Fuc orα1,4-Fuc. 511 was reduced by about 30%, indicating that the signalcontained a minor component with α1,2-Fuc, preferentially includingFucα2Galβ4Glc (Fucα2′Lac, 2′-fucosyllactose).

When the α1,3/4-fucosidase reaction product was further digested withα1,2-fucosidase, 876 was completely digested into 730, indicating thatthe structure of the majority of the signal intensity containednon-reducing terminal α1,2-Fuc, preferably including the structureFucα2[Hex₁HexNAc₁]Lac, more preferably including Fucα2GalHexNAcLac.Another partly digested glycan signal was Hex₄HexNAc₂dHex₁ (1241) thatwas thus indicated to contain α1,2-Fuc, preferably including thestructure Fucα2-[Hex₂HexNAc₂]Lac, more preferably includingFucα2Gal[Hex₁HexNAc₂]Lac. 511 was completely digested, indicating thatthe original signal contained a major component with α1,3/4-Fuc,preferentially including Galβ4(Fucα3)Glc (3-fucosyllactose).

When the α1,3/4-fucosidase and α1,2-fucosidase reaction product wasfurther digested with β1,4-galactosidase, the majority of the newlyformed 730 was not digested, i.e. the relative proportion of 568 was notincreased compared to β1,4-galactosidase digestion without precedingfucosidase treatments. This indicated that the majority of 876 did notcontain β1,4-Gal subterminal to Fuc. Further, 892 was not digested,indicating that it did not contain non-reducing terminal β1,4-Gal.

When the α1,3/4-fucosidase, α1,2-fucosidase, and β1,4-galactosidasereaction product was further digested with β1,3-galactosidase, thesignal intensity of 892 was reduced, indicating that it included glycanswith terminal β1,3-Gal. The signal intensity of 568 was increasedrelative to 730, indicating that also 730 included glycans with terminalβ1,3-Gal.

The experimental structures of the major hESC glycosphingolipid neutralglycan signals were thus determined (‘>’ indicates the order ofpreference among the lipid glycan structures of hESC; ‘[ ]’ indicatesthat the oligosaccharide sequence in brackets may be either branched orunbranched; ‘( )’ indicates a branch in the structure):

730 Hex₃HexNAc₁ > Hex₁HexNAc₁Lac > Galβ4GlcNAcLac 876 Hex₃HexNAc₁dHex₁ >Fucα2[Hex₁HecNAc₁]Lac > Fucα2Galβ4GlcNAcLac > Fucα3/4[Hex₁HecNAc₁]Lac568 Hex₂HexNAc₁ > HecNAcLac 933 Hex₃HexNAc₂ > [Hex₁HecNAc₂]Lac 892Hex₄HexNAc₁ > [Hex₂HecNAc₁]Lac > Galβ3[Hex₁HecNAc₁]Lac 1095Hex₄HexNAc₂ > [Hex₂HecNAc₂]Lac > Galβ3HexNAc[Hex₁HecNAc₁]Lac >Galβ4GlcNAc[Hex₁HecNAc₁]Lac 1460 Hex₅HexNAc₃ > [Hex₃HecNAc₃]Lac >Galβ4GlcNAc(Galβ4GlcNAc)[Hex₁HecNAc₁]Lac

Acidic lipid glycans. The analyzed mass spectrometric profile of thehESC glycosphingolipid sialylated glycan fraction is shown in FIG. 46.The four major, glycan signals, together comprising more than 96% of thetotal glycan signal intensity, corresponded to monosaccharidecompositions NeuAc₁Hex₃HexNAc₁ (997), NeuAc₁Hex₂HexNAc₁ (835),NeuAc₁Hex₄HexNAc₁ (1159), and NeuAc₂Hex₃HexNAc₁ (1288).

The acidic glycan fraction was subjected to α2,3-sialidase digestion andthe resulting neutral and acidic glycan fractions were purified andanalyzed separately. In the acidic fraction, signals 1159 and 1288 weredigested and 835 was partly digested. In the neutral fraction, signals730 and 892 were the major appeared signals. These results indicatedthat: 1159 consisted mainly of glycans with α2,3-NeuAc, 1288 containedat least one α2,3-NeuAc, a major proportion of glycans in 835 containedα2,3-NeuAc, and in the original sample a major proportion ofNeuAc₁₋₂Hex₃HexNAc₁ contained solely α2,3-linked NeuAc.

Human Mesenchymal Stem Cells (MSC)

Bone marrow derived (BM) MSC neutral lipid glycans. The analyzed massspectrometric profile of the BM MSC glycosphingolipid neutral glycanfraction is shown in FIG. 45. The six major glycan signals, togethercomprising more than 94% of the total glycan signal intensity,corresponded to monosaccharide compositions Hex₃HexNAc₁ (730),Hex₂HexNAc₁ (568), Hex₂dHex₁ (511), Hex₂HexNAc₂dHex₂ (1063),Hex₃HexNAc₂dHex₂ (1225), and Hex₃HexNAc₂dHex₁ (1079). The four mostabundant signals (730, 568, 511, and 1063) together comprised more than75% of the total intensity.

Cord blood derived (CB) MSC neutral lipid glycans. The analyzed massspectrometric profile of the CB MSC glycosphingolipid neutral glycanfraction is shown in FIG. 45. The ten major glycan signals, togethercomprising more than 92% of the total glycan signal intensity,corresponded to monosaccharide compositions Hex₂HexNAc₁ (568),Hex₃HexNAc₁ (730), Hex₄HexNAc₂ (1095), Hex₅HexNAc₃ (1460), Hex₃HexNAc₂(933), Hex₂dHex₁ (511), Hex₂HexNAc₂dHex₂ (1063), Hex₄HexNAc₃ (1298),Hex₃HexNAc₂dHex₂ (1225), and Hex₂HexNAc₂ (771). The five most abundantsignals (568, 730, 1095, 1460, and 933) together comprised more than 82%of the total intensity.

In β1,4-galactosidase digestion, the relative signal intensities of1095, 1460, and 730 were reduced by about 90%, 95%, and 20%,respectively. This suggests that CB MSC contained major glycancomponents with non-reducing terminal β1,4-Gal epitopes, preferablyincluding the structures Galβ4GlcNAcβ[Hex₁HexNAc₁]Lac,Galβ4GlcNAc[Hex₂HexNAc₂]Lac, and Galβ4GlcNAcLac. Further, the glycansignal Hex₅HexNAc₃ (1460) was digested into Hex₄HexNAc₃ (1298) andmostly into Hex₃HexNAc₃ (1136), indicating that the original signalcontained glycan structures containing either one or two β1,4-Gal, andthat the majority of the original glycans contained two β1,4-Gal,preferentially including the structureGalβ4GlcNAc(Galβ4GlcNAc)[Hex₁HexNAc₁]Lac. Similarly, 1095 was digestedinto Hex₂HexNAc₂ (771) in addition to 933, indicating that the originalsignal contained glycan structures containing either one or twoβ1,4-Gal, and that the minority of the original glycans contained twoβ1,4-Gal, preferentially including the structureGalβ4GlcNAc(Galβ4GlcNAc)Lac.

The experimental structures of the major CB MSC glycosphingolipidneutral glycan signals were thus determined (‘>’ indicates the order ofpreference among the lipid glycan structures of hESC; ‘[ ]’ indicatesthat the oligosaccharide sequence in brackets may be either branched orunbranched; ‘( )’ indicates a branch in the structure):

568 Hex₂HexNAc₁ > HecNAcLac 730 Hex₃HexNAc₁ > Hex₁HexNAc₁Lac >Galβ4GlcNAcLac 1095 Hex₄HexNAc₂ > [Hex₂HecNAc₂]Lac >Galβ4GlcNAc[Hex₁HecNAc₁]Lac > Galβ4GlcNAc(Galβ4GlcNAc)Lac 1460Hex₅HexNAc₃ > [Hex₃HecNAc₃]Lac > Galβ4GlcNAc[Hex₂HecNAc₂]Lac >Galβ4GlcNAc(Galβ4GlcNAc)[Hex₁HecNAc₁]Lac 933 Hex₃HexNAc₂ >Hex₁HexNAc₂Lac

Sialylated lipid glycans. The analyzed mass spectrometric profile of thehESC glycosphingolipid sialylated glycan fraction is shown in FIG. 46.The five major glycan signals of BM MSC, together comprising more than96% of the total glycan signal intensity, corresponded to monosaccharidecompositions NeuAc₁Hex₂HexNAc₁ (835), NeuAc₁Hex₁HexNAc₁dHex₁ (819),NeuAc₁Hex₃HexNAc₁ (997), NeuAc₁Hex₃HexNAc₁dHex₁ (1143), andNeuAc₂Hex₁HexNAc₂dHex₁ (1313). The six major glycan signals of CB MSC,together comprising more than 92% of the total glycan signal intensity,corresponded to monosaccharide compositions NeuAc₁Hex₂HexNAc₁ (835),NeuAc₁Hex₃HexNAc₁ (997), NeuAc₂Hex₂ (905), NeuAc₁Hex₄HexNAc₂ (1362),NeuAc₁Hex₅HexNAc₃ (1727), and NeuAc₂Hex₂HexNAc₁ (1126).

Human Cord Blood Mononuclear Cells (CB MNC)

CB MNC neutral lipid glycans. The analyzed mass spectrometric profile ofthe CB MNC glycosphingolipid neutral glycan fraction is shown in FIG.45. The five major glycan signals, together comprising more than 91% ofthe total glycan signal intensity, corresponded to monosaccharidecompositions Hex₃HexNAc₁ (730), Hex₂HexNAc₁ (568), Hex₃HexNAc₁dHex₁(876), Hex₄HexNAc₂ (1095), and Hex₄HexNAc₂dHex₁ (1241).

In β1,4-galactosidase digestion, the relative signal intensities of 730and 1095 were reduced by about 50% and 90%, respectively. This suggeststhat the signals contained major components with non-reducing terminalβ1,4-Gal epitopes, preferably including the structures Galβ4GlcNAcβLacand Galβ4GlcNAcβ[Hex₁HexNAc₁]Lac. Further, the glycan signal Hex₅HexNAc₃(1460) was digested to Hex4HexNAc₃ (1298) and Hex₃HexNAc₃ (1136),indicating that the original signal contained glycan structurescontaining either one or two β1,4-Gal.

The experimental structures of the major CB MNC glycosphingolipidneutral glycan signals were thus determined (‘>’ indicates the order ofpreference among the lipid glycan structures of hESC; ‘[ ]’ indicatesthat the oligosaccharide sequence in brackets may be either branched orunbranched; ‘( )’ indicates a branch in the structure):

730 Hex₃HexNAc₁ > Hex₁HexNAc₁Lac > Galβ4GlcNAcLac 568 Hex₂HexNAc₁ >HecNAcLac 876 Hex₃HexNAc₁dHex₁ > [Hex₁HecNAc₁dHex₁]Lac >Fuc[Hex₁HecNAc₁]Lac 1095 Hex₄HexNAc₂ > [Hex₂HecNAc₂]Lac >Galβ4GlcNAc[Hex₁HecNAc₁]Lac 1241 Hex₄HexNAc₂dHex₁ >[Hex₂HecNAc₂dHex₁]Lac > Fuc[Hex₂HecNAc₂]Lac 1460 Hex₅HexNAc₃ >[Hex₃HecNAc₃]Lac > Galβ4GlcNAc[Hex₂HecNAc₂]Lac >Galβ4GlcNAc(Galβ4GlcNAc)[Hex₁HecNAc₁]Lac

Sialylated lipid glycans. The analyzed mass spectrometric profile of theCB MNC glycosphingolipid sialylated glycan fraction is shown in FIG. 46.The three major glycan signals of CB MNC, together comprising more than96% of the total glycan signal intensity, corresponded to monosaccharidecompositions NeuAc₁Hex₃HexNAc₁ (997), NeuAc₁Hex₄HexNAc₂ (1362), andNeuAc₁Hex₅HexNAc₃ (1727).

Overview of Human Stem Cell Glycosphingolipid Glycan Profiles

The neutral glycanfractions of all the present sample types altogethercomprised 45 glycan signals. The proposed monosaccharide compositions ofthe signals were composed of 2-7 Hex, 0-5 HexNAc, and 0-4 dHex. Glycansignals were detected at monoisotopic m/z values between 511 and 2263(for [M+Na]⁺ ion).

Major neutral glycan signals common to all the sample types were 730,568, 1095, and 933, corresponding to the glycan structure groupsHex₀₋₁HexNAc₁Lac (568 or 730) and Hex₁₋₂HexNAc₂Lac (933 or 1095), ofwhich the former glycans were more abundant and the latter lessabundant. A general formula of these common glycans isHex_(m)HexNAc_(n)Lac, wherein m is either n or n−1, and n is either 1 or2.

Neutral Glycolipid Profiles of Human Stem Cell Types:

Glycan signals typical to hESC preferentially include 876 and 892(especially compared to MSC); the former preferentially corresponds toFucHexHexNAcLac, wherein α1,2-Fuc is preferential to α1,3/4-Fuc, and thelatter preferentially corresponds to Hex₂HexNAcLac, and morepreferentially to Galβ3[Hex₁HexNAc₁]Lac; the glycan core compositionHex₄HexNAc₁ was especially characteristic of hESC compared to otherhuman stem cell types, in addition to fucosylation and morepreferentially α1,2-linked fucosylation.

Glycan signals typical to both CB and BM MSC preferentially include 771,1063, 1225; more preferentially including compositionsdHex_(0/2)Hex₀₋₁HexNAc₂Lac.

Glycan signals typical to especially BM MSC preferentially include 511and fucosylated structures, preferentially multifucosylated structures.

Glycan signals typical to especially CB MSC preferentially include 1460and 1298, as well as large neutral glycolipids, especiallyHex₂₋₃HexNAc₃Lac. In addition, low fucosylation and/or high expressionof terminal β1,4-Gal was typical to especially CB MSC.

Glycan signals typical to CB MNC preferentially include compositionsdHex₀₋₁[HexHexNAc]₁₋₂Lac, more preferentially high relative amounts of730 compared to other signals; and fucosylated structures; and glycanprofiles with less variability and/or complexity than other stem celltypes.

The acidic glycan fractions of all the present sample types altogethercomprised 38 glycan signals. The proposed monosaccharide compositions ofthe signals were composed of 0-2 NeuAc, 2-9 Hex, 0-6 HexNAc, 0-3 dHex,and/or 0-1 sulphate or phosphate esters. Glycan signals were detected atmonoisotopic m/z values between 786 and 2781 (for [M-H]⁻ ion).

The acidic glycosphingolipid glycans of CB MNC were mainly composed ofNeuAc₁Hex_(n+2)HexNAc_(n), wherein 1≦n≦3, indicating that theirstructures were NeuAc₁ [HexHexNAc]₁₋₃Lac.

Terminal glycan epitopes that were demonstrated in the presentexperiments in stem cell glycosphingolipid glycans include:

Gal Galβ4Glc (Lac)

Galβ4GlcNAc (LacNAc type 2)

Galβb 3

Non-reducing terminal HexNAc

Fuc α1,2-Fuc α1,3-Fuc Fucα2Gal

Fucα2Galβ4GlcNAc (H type 2)Fucα2Galβ4Glc (2′-fucosyllactose)

Fucα3GlcNAc Galβ4(Fucα3)GlcNAc (Lex) Fucα3Glc

Galβ4(Fucα3)Glc (3-fucosyllactose)

Neu5Ac Neu5Acα2,3 Neu5Acα2,6

Development-related glycan epitope expression. According to the presentinvention, the glycosphingolipid glycan composition Hex₄HexNAc₁preferentially corresponds to (iso)globo structures. The glycan sequenceof the SSEA-3 glycolipid antigen has been determined to beGalβ3GalNAcβ3Galα4Galβ4Glc, which corresponds to the glycan signalHex₄HexNAc₁ (892) detected in the present experiments only in hESC.Similarly, the glycan sequence of the SSEA-4 glycolipid antigen has beendetermined to be NeuAcα3Galβ3GalNAcβ3Galα4Galβ4Glc, which corresponds tothe glycan signal NeuAc₁Hex₄HexNAc₁ (1159) detected in the presentexperiments only in hESC. Consistent with the present glycan structureanalyses, the hESC samples were determined to be SSEA-3 and SSEA-4positive by monoclonal antibody staining as described in the precedingExamples. In contrast to mouse ES cells, hESC do not express the SSEA-1antigen; consistent with this we found only low expression levels ofα1,3/4-fucosylated neutral glycolipid glycans. In contrast, we were ableto show that the major fucosylated structures of hESC glycosphingolipidglycans contain α1,2-Fuc, which is a molecular level explanation to themouse-human difference in SSEA-1 reactivity.

Example 31 Stem Cell O-glycan Structural Analysis Results and Discussion

Total de-N-glycosylated protein pool of the hESC line FES 29, which wasalready treated with N-glycosidase F to get rid of N-glycans, wassubjected to non-reductive β-elimination to harvest the total hESCO-glycan pool as described in the preceding Examples. The liberatedglycans were purified, divided into neutral and acidic fractions, andanalyzed by MALDI-TOF mass spectrometry as described.

Structural analysis of the major neutral O-glycans. The two major[M+Na]⁺ glycan signals emerging from the O-glycan pool were m/z 771(Hex2HexNAc2) and 917 (Hex2HexNAc2dHex1). O-glycans were then treatedwith β1,4-galactosidase as described in the preceding Examples. The m/z771 glycan signal was sensitive to this treatment, indicating that thecorresponding hESC neutral O-glycans had preferentially containednon-reducing terminal β1,4-linked Gal.

Structural analysis of the major acidic O-glycans. The five major [M-H]⁻glycan signals emerging from the O-glycan pool were 964.35(NeuAc2HexHexNAc), 1038.49 (NeuAc1Hex2HexNAc2), 1329.56(NeuAc2Hex2HexNAc2), 1403.62 (NeuAc1Hex3HexNAc3), and 1768.75(NeuAc1Hex4HexNAc4).

O-glycans were then treated with α2,3-sialidase as described in thepreceding Examples. All these major peaks were absent in the massspectrum recorded after this treatment. The loss of this glycan seriesconsisting of sialic acid with varying number of HexHexNAc disaccharideindicated that the corresponding hESC acidic O-glycans had containedpreferentially α2,3-linked sialic acids. In addition, the signal at m/z1329.56 containing two sialic acids disappeared, indicating that bothsialic acids were preferentially α2,3-linked.

The substrate specificity of α2,3-sialidase was tested in parallelexperiments using two synthetic oligosaccharides, namelyNeuAcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc andNeuAcα2,6[Galβ1,4GlcNAβ1,3(Galβ1,4GlcNAcβ1,6)]Galβ1,4Glc. The enzymespecifically hydrolyzed α2,3-linked sialic acids and left α2,6-linkedsialic acids intact.

Example 32 Lectin Based Selection of CB MNC Cell Populations

The FACS experiments with fluorescein-labeled lectins and CB MNC wereperformed essentially similarly to Example 20. Double stainings wereperformed with CD34 specific monoclonal antibody (Jaatinen et al., 2006)with complementary fluorescent dye. Erythroblast depletion from CD MNCfraction was performed by anti-glycophorin A (GlyA) monoclonal antibodynegative selection.

Results and Discussion

Compared to the CB MNC fraction, GlyA depleted CB MNC showed decreasedstaining in FACS with the following lectins (the decrease in % inparenthesis): PWA (48%), LTA (59%), UEA (34%), STA, MAA, and PNA (alllatter three less than 23%); indicating that GlyA depletion increasedthe resolving power of the lectins in cell sorting.

In FACS double staining with both fluorescein-labeled lectins andanti-CD34 antibody, the following lectins colocalized with CD34+ cells:STA (3/3 samples), HHA (3/3 samples), PSA (3/3 samples), RCA (3/3samples), and partly also NPA (2/3 samples). In contrast, the followinglectins did not colocalize with CD34+ cells: GNA (3/3 samples) and PWA(3/3 samples), and partly also LTA (2/3 samples), WFA (2/3 samples), andGS-II (2/3 samples).

Taken together with the results of Example 21, the present resultsindicate that lectins can enrich CD34+ cells from CB MNC by bothnegative and positive selection, for example:

-   -   1) GNA binds to about 70% of CB MNC but not to CD34+ cells,        leading to about 3× enrichment in negative selection of CB MNC        in CD34+ cell isolation.    -   2) STA binds to about 50% of CB MNC and also to CD34+ cells,        leading to about 2× enrichment in positive selection of CB MNC        in CD34+ cell isolation.    -   3) UEA binds to about 50% of CB MNC and also to CD34+ cells,        leading to about 2× enrichment in positive selection of CB MNC        in CD34+ cell isolation.

Example 33 Galectin Gene Expression Profiles of Stem Cells EXPERIMENTALPROCEDURES

Gene expression analysis of CB CD133+ cells has been described (Jaatinenet al., 2006) and the present analysis was performed essentiallysimilarly. The galectins whose gene expression profile was analyzedincluded (corresponding Affymetrix codes in parenthesis): Galectin-1(201105_at), galectin-2 (208450_at), galectin-3 (208949_s_at),galectin-4 (204272_at), galectin-6 (200923_at), galectin-7 (206400_at),galectin-8 (208933_sat), galectin-9 (203236_s_at), galectin-10(206207_at), galectin-13 (220158_at).

Results and Discussion

In CB CD133+ versus CD133−, as well as CD34+ versus CD34− CB MNC cells,the galectin gene expression profile was as follows: Overall, galectins1, 2, 3, 6, 8, 9, and 10 showed gene expression in both CD34+/CD133+cells. Galectins 1, 2, and 3 were downregulated in both CD34+/CD133+cells with respect to CD34−/CD133− cells, and in addition galectin 10was downregulated in CD133+ cells with respect to CD133− cells. Incontrast, in both CD34+/CD133+ cells galectin 8 was upregulated withrespect to CD34−/CD133− cells.

In hESC versus EB samples, the galectin gene expression profile was asfollows: Overall, galectins 1, 3, 6, 8, and 13 showed gene expression inhESC. Galectin 3 was clearly downregulated with respect to EB, and inaddition galectin 13 was downregulated in 2 out of 4 hESC lines. Incontrast, galectin 1 was clearly upregulated in all hESC lines.

The results indicate that both CB CD34+/CD133+ stem cell populations andhESC have an interesting and distinct galectin expression profiles,leading to different galectin ligand affinity profiles (Hirabayashi etal., 2002). The results further correlate with the glycan analysisresults showing abundant galectin ligand expression in these stem cells,especially non-reducing terminal β-Gal and type II LacNAc, poly-LacNAc,β1,6-branchedpoly-LacNAc, and complex-type N-glycan expression.

TABLE 1 Preferred neutral glycan compositions. Calculated mass-to-chargeratios (calc. m/z) refer to the first isotope signal of [M + Na]⁺ ion.Proposed composition calc. m/z HexHexNAc 406.13 Hex3 527.16HexHexNAcdHex 552.19 Hex2HexNAc 568.19 HexHexNAc2 609.21 Hex4 689.21Hex2HexNAcdHex 714.24 Hex3HexNAc 730.24 HexHexNAc2dHex 755.27Hex2HexNAc2 771.26 HexHexNAc3 812.29 Hex5 851.26 Hex2HexNAcdHex2 860.30Hex4HexNAc 892.29 HexHexNAc2dHex2 901.33 Hex2HexNAc2dHex 917.32Hex3HexNAc2 933.32 HexHexNAc3dHex 958.35 Hex2HexNAc3 974.34Hex2HexNAcdHex3 1006.36 Hex6 1013.32 HexHexNAc4 1015.37 Hex3HexNAcdHex21022.35 Hex5HexNAc 1054.34 Hex2HexNAc2dHex2 1063.38 Hex3HexNAc2dHex1079.38 Hex4HexNAc2 1095.37 HexHexNAc3dHex2 1104.41 Hex2HexNAc3dHex1120.40 Hex3HexNAc3 1136.40 Hex2HexNAcdHex4 1152.42 HexHexNAc4dHex1161.43 Hex7 1175.37 Hex2HexNAc4 1177.42 Hex2HexNAc2dHex3 1209.44Hex6HexNAc 1216.40 HexHexNAc5 1218.45 Hex3HexNAc2dHex2 1225.43Hex4HexNAc2dHex 1241.43 Hex5HexNAc2 1257.42 Hex2HexNAc3dHex2 1266.46Hex3HexNAc3dHex 1282.45 Hex4HexNAc3 1298.45 HexHexNAc4dHex2 1307.49Hex2HexNAc4dHex 1323.48 Hex8 1337.42 Hex3HexNAc4 1339.48Hex2HexNAc2dHex4 1355.50 HexHexNAc5dHex 1364.51 Hex3HexNAc2dHex3 1371.49Hex7HexNAc 1378.45 Hex4HexNAc2dHex2 1387.49 Hex2HexNAc5 1380.50Hex5NexNAc2dHex 1403.48 Hex2HexNAc3dHex3 1412.52 Hex6HexNAc2 1419.48HexHexNAc6 1421.53 Hex3HexNAc3dHex2 1428.51 Hex4HexNAc3dHex 1444.51HexHexNAc4dHex3 1453.54 Hex5HexNAc3 1460.50 Hex2HexNAc4dHex2 1469.54Hex3HexNAc4dHex 1485.53 Hex9 1499.48 Hex4HexNAc4 1501.53 HexHexNAc5dHex21510.57 Hex3HexNAc2dHex4 1517.55 Hex2HexNAc5dHex 1526.56Hex4HexNAc2dHex3 1533.54 Hex8HexNAc 1540.50 Hex3HexNAc5 1542.56Hex5HexNAc2dHex2 1549.54 Hex6HexNAc2dHex 1565.53 Hex3HexNAc3dHex31574.57 Hex7HexNAc2 1581.53 Hex2HexNAc6 1583.58 Hex4HexNAc3dHex2 1590.57Hex5HexNAc3dHex 1606.56 Hex2HexNAc4dHex3 1615.60 Hex6HexNAc3 1622.56Hex3HexNAc4dHex2 1631.59 Hex4HexNAc4dHex 1647.59 Hex10 1661.53Hex5HexNAc4 1663.58 Hex2HexNAc5dHex2 1672.62 Hex3HexNAc5dHex 1688.61Hex5HexNAc2dHex3 1695.60 Hex9HexNAc 1702.56 Hex4HexNAx5 1704.61Hex6HexNAc2dHex2 1711.59 Hex3HexNAc3dHex4 1720.63 Hex7HexNAc2dHex1727.59 Hex2HexNAc6dHex 1729.64 Hex4HexNAc3dHex3 1736.62 Hex8HexNAc21743.58 Hex3HexNAc6 1745.64 Hex5HexNAc3dHex2 1752.62 Hex6HexNAc3dHex1768.61 Hex3HexNAc4dHex3 1777.65 Hex7HexNAc3 1784.61 Hex4HexNAc4dHex21793.64 Hex5HexNAc4dHex 1809.64 Hex2HexNAc5dHex3 1818.68 Hex11 1823.58Hex6HexNAc4 1825.63 Hex3HexNAc5dHex2 1834.67 Hex4HexNAc5dHex 1850.67Hex6HexNAc2dHex3 1857.65 Hex10HexNAc 1864.61 Hex5HexNAc5 1866.66Hex7HexNAc2dHex2 1873.64 Hex2HexNAc6dHex2 1875.70 Hex4HexNAc3dHex41882.68 Hex8HexNAc2dHex 1889.64 Hex3HexNAc6dHex 1891.69 Hex5HexNAc3dHex31898.68 Hex9HexNAc2 1905.63 Hex4HexNAc6 1907.69 Hex6HexNAc3dHex2 1914.67Hex3HexNAc4dHex4 1923.71 Hex7HexNAc3dHex 1930.67 Hex2HexNAc7dHex 1932.72Hex4HexNAc4dHex3 1939.70 Hex8HexNAc3 1946.66 Hex5HexNAc4dHex2 1955.70Hex6HexNAc4dHex 1971.69 Hex3HexNAc5dHex3 1980.73 Hex12 1985.63Hex7HexNAc4 1987.69 Hex4HexNAc5dHex2 1996.72 Hex5HexNAc5dHex 2012.72Hex7HexNAc2dHex3 2019.70 Hex2HexNAc6dHex3 2021.76 Hex11HexNAc 2026.66Hex6HexNAc5 2028.71 Hex8HexNAc2dHex2 2035.70 Hex3HexNAc6dHex2 2037.75Hex5HexNAc3dHex4 2044.73 Hex4HexNAc6dHex 2053.75 Hex6HexNAc3dHex32060.73 Hex10HexNAc2 2067.69 Hex5HexNAc6 2069.74 Hex7HexNAc3dHex22076.72 Hex2HexNAc7dHex2 2078.78 Hex4HexNAc4dHex4 2085.76Hex8HexNAc3dHex 2092.72 Hex3HexNAc7dHex 2094.77 Hex5HexNAc4dHex3 2101.76Hex9HexNAc3 2108.71 Hex4HexNAc7 2110.77 Hex6HexNAc4dHex2 2117.75Hex3HexNAc5dHex4 2126.79 Hex7HexNAc4dHex 2133.75 Hex4HexNAc5dHex32142.78 Hex13 2147.69 Hex8HexNAc4 2149.74 Hex5HexNAc5dHex2 2158.78Hex6HexNAc5dHex 2174.77 Hex8HexNAc2dHex3 2181.76 Hex3HexNAc6dHex32183.81 Hex12HexNac 2188.71 Hex7HexNAc5 2190.77 Hex4HexNAc6dHex2 2199.80Hex5HexNAc6dHex 2215.80 Hex7HexNAc3dHex3 2222.78 Hex2HexNAc7dHex32224.84 Hex11HexNAc2 2229.74 Hex6HexNAc6 2231.79 Hex8HexNAc3dHex22238.78 Hex3HexNAc7dHex2 2240.83 Hex5HexNAc4dHex4 2247.81Hex4HexNAc7dHex 2256.83 Hex6HexNAc4dHex3 2263.81 Hex5HexNAc7 2272.82Hex7HexNAc4dHex2 2279.80 Hex4HexNAc5dHex4 2288.84 Hex5HexNAc5dHex32304.84 Hex14 2309.74 Hex9HexNAc4 2311.79 Hex6HexNAc5dHex2 2320.83Hex7HexNAc5dHex 2336.82 Hex4HexNAc6dHex3 2345.86 Hex8HexNAc5 2352.82Hex5HexNAc6dHex2 2361.86 Hex6HexNAc6dHex 2377.85 Hex8HexNAc3dHex32384.83 Hex3HexNAc7dHex3 2386.89 Hex12HexNac2 2391.79 Hex7HexNAc62393.85 Hex4HexNAc7dHex2 2402.88 Hex6HexNAc4dHex4 2409.87Hex5HexNAc7dHex 2418.88 Hex7HexNAc4dHex3 2425.86 Hex6HexNAc7 2434.87Hex5HexNAc5dHex4 2450.89 Hex6HexNAc5dHex3 2466.89 Hex15 2471.79Hex7HexNAc5dHex2 2482.88 Hex8HexNAc5dHex 2498.88 Hex5HexNAc6dHex32507.91 Hex6HexNAc6dHex2 2523.91 Hex7HexNAc6dHex 2539.90Hex4HexNAc7dHex3 2548.94 Hex13HexNAc2 2553.85 Hex8HexNAc6 2555.90Hex5HexNAc7dHex2 2564.94 Hex6HexNAc7dHex 2580.93 Hex6HexNAc5dHex42612.95 Hex7HexNAc5dHex3 2628.94 Hex16 2633.85 Hex8HexNAc5dHex2 2644.94Hex6HexNAc6dHex3 2669.97 Hex7HexNAc6dHex2 2685.96 Hex5HexNAc7dHex32710.99 Hex14HexNAc2 2715.90 Hex6HexNAc7dHex2 2726.99 Hex7HexNAc7dHex2742.98 Hex8HexNAc7 2758.98 Hex7Hexnac5dHex4 2775.00 Hex8HexNAc5dHex32790.99 Hex17 2795.90 Hex7HexNAc6dHex3 2832.02 Hex16HexNAc 2836.92Hex9HexNAc6dHex 2864.01 Hex6HexNAc7dHex3 2873.05 Hex15HexNAc2 2877.95Hex8HexNAc7dHex 2905.04 Hex8Hexnac5dHex4 2937.05 Hex18 2957.95Hex7HexNAc6dHex4 2978.08 Hex17HexNAc 2998.98 Hex8HexNAc7dHex2 3051.09Hex9HexNAc8 3124.11 Hex8HexNAc6dHex4 3140.13 Hex8HexNAc7dHex3 3197.15Hex9HexNAc8dHex/ 3270.17 Hex7HexNAc6dHex6 Hex9HexNAc6dHex4 3302.18Hex8HexNAc7dHex4 3343.21 Hex9HexNAc8dHex2 3416.23 Hex10HexNAc6dHex43464.24 Hex10HexNAc9 3489.24 Hex9HexNAc8dHex3 3562.28 Hex11HexNAc6dHex43626.29 Hex10HexNAc9dHex 3635.30 Hex9HexNAc8dHex4 3708.34Hex10HexNAc9dHex2/ 3781.36 Hex8HexNAc7dHex7 Hex9HexNAc8dHex5/ 3854.40Hex7HexNAc6dHex10

TABLE 2 Preferred acidic glycan compositions. Calculated mass-to-chargeratios (calc. m/z) refer to the first isotope signal of [M − H]⁻ ion.Proposed composition calc. m/z NeuAcHexHexNAc 673.23 NeuAcHexHexNAcdHex819.29 NeuAcHex2HexNAc 835.28 NeuAcHexHexNAc2 876.31 NeuAc2HexHexNAc964.33 NeuAcHexHexNAcdHex2 965.35 NeuAcHex2HexNAcdHex 981.34Hex3HexNAc2SP 989.28 NeuAcHex3HexNAc 997.34 NeuAcHexHexNAc2dHex 1022.37NeuAcHex2HexNAc2 1038.36 NeuAcHexHexNAc3 1079.39 NeuAc2HexHexNAcdHex1110.38 NeuAc2Hex2HexNAc 1126.38 NeuAcHex2HexNAcdHex2 1127.40NeuAcHex3HexNAcdHex 1143.39 Hex4HexNAc2SP 1151.33 NeuAcHex4HexNAc1159.39 NeuAc2HexHexNAc2 1167.41 NeuAcHexHexNAc2dHex2 1168.43NeuAcHex2HexNAc2dHex 1184.42 Hex3HexNAc3SP 1192.36 NeuAcHex3HexNAc2/1200.42 NeuGcHex2HexNAc2dHex NeuGcHex3HexNAc2 1216.41NeuAcHexHexNAc3dHex 1225.45 NeuAcHex2HexNAc3 1241.44NeuAc2Hex2HexNAcdHex 1272.44 NeuAcHexHexNAc4 1282.47 NeuAc2Hex3HexNAc1288.43 NeuAcHex4HexNAcdHex 1305.45 NeuAc2HexHexNAc2dHex 1313.46NeuAcHex5HexNAc/ 1321.44/ NeuAcHex2HexNAc3SP 1321.40 NeuAc2Hex2HexNAc2/1329.46 NeuGcNeuAcHexHexNAc2dHex NeuAcHex2HexNAc2dHex2 1330.48Hex3HexNAc3dHexSP 1338.41 NeuAcHex3HexNAc2dHex 1346.47 Hex4HexNAc3SP1354.41 NeuAcHex4HexNAc2 1362.47 NeuAc2HexHexNAc3 1370.48NeuAcHex2HexNAc3dHex 1387.50 NeuAcHex3HexNAc3 1403.49 NeuGcHex3HexNAc31419.49 NeuAcHexHexNAc4dHex 1428.53 NeuAc2Hex3HexNAcdHex 1434.49NeuAcHex2HexNAc4 1444.52 NeuAcHex3HexNAc3Ac 1445.51 NeuAc2Hex4HexNAc1450.48 Hex5HexNAc2dHexSP 1459.44 NeuAc2Hex2HexNAc2dHex 1475.52NeuAcHex6HexNAc/ 1483.49/ NeuAcHex3HexNAc3SP 1483.45 NeuAc2Hex3HexNAc21491.51 NeuAcHex3HexNAc2dHex2 1492.53 Hex4HexNAc3dHexSP 1500.47NeuAcHex4HexNAc2dHex 1508.53 NeuAc2HexHexNAc3dHex/ 1516.54/Hex5HexNAc3SP 1516.46 NeuAcHex5HexNAc2 1524.52 NeuAc2Hex2HexNAc3 1532.54NeuAcHex2HexNAc3dHex2 1533.56 NeuAcHex3HexNAc3dHex 1549.55NeuAc2Hex2HexNAc2dHexSP 1555.47 Hex4HexNAc4SP 1557.49NeuAcHex3HexNAc3(SP)2 1563.41 NeuAcHex4HexNAc3 1565.55 NeuAc2HexHexNAc41573.56 NeuGcHex4HexNAc3 1581.54 NeuAcHex2HexNac4dHex 1590.58NeuAc2Hex4HexNAcdHex 1596.54 NeuAcHex3HexNAc4 1606.57NeuAc2Hex2HexNAc2dHex2/ 1621.57/ Hex6HexNAc2dHexSP 1621.49NeuAc2Hex3HexNAc2dHex 1637.57 NeuAcHex4HexNAc3SP 1645.50NeuAcHex2HexNAc5 1647.60 NeuAcHex4HexNAc2dHex2 1654.58 Hex5HexNAc3dHexSP1662.52 NeuAcHex5HexNAc2dHex 1670.58 NeuAc2Hex2HexNAc3dHex 1678.60NeuAcHex2HexNAc3dHex3 1679.62 NeuAcHex6HexNAc2 1686.57 NeuAc2Hex3HexNAc31694.59 Hex4HexNAc4dHexSP 1703.55 NeuAcHex3HexNAc3dHex(SP)2 1709.47NeuGcNeuAcHex3HexNAc3 1710.59 NeuAcHex4HexNAc3dHex 1711.61 Hex5HexNAc4SP1719.54 NeuAcHex4HexNAc3(SP)2 1725.46 Hex4HexNAc3dHex2(SP)2/ 1726.48/NeuGc2Hex3HexNAc3 1726.58 NeuAcHex5HexNAc3/ 1727.60 NeuGcHex4HexNAc3dHexNeuAc2Hex2HexNAc4 1735.62 NeuAcHex2HexNAc4dHex2 1736.64 NeuGcHex5HexNAc31743.60 NeuAcHex3HexNAc4dHex 1752.63 NeuAc2Hex2HexNAc3dHexSP 1758.55NeuAcHex3HexNAc4(SP)2/ 1766.49/ NeuAcHex6HexNAc2SP 1766.53Hex6HexNAc2dHex2SP/ 1767.55/ Hex3HexNAc4dHex2(SP)2/ 1767.51NeuAc2Hex2HexNAc2dHex3 NeuAcHex4HexNAc4 1768.63 NeuAc2Hex6HexNAc/1774.59/ NeuAc2Hex3HexNAc3SP 1774.55 Hex7HexNAc2dHexSP 1783.55NeuGcHex4HexNac4 1784.62 NeuAcHex4HexNAc3dHexSP 1791.56NeuAcHex2HexNAc5dHex 1793.66 NeuAc2Hex4HexNAc2dHex/ 1799.62Hex5HexNAc4(SP)2 NeuAcHex3HexNac5 1809.65 NeuAc2Hex5HexNAc2/ 1815.62NeuAc2Hex2HexNAc4SP NeuAcHex5HexNAc2dHex2/ 1816.64NeuAcHex2HexNAc4dHex2SP Hex6NexNAc3dHexSP 1824.57 NeuGcHex3HexNAc51825.65 NeuAcHex6HexNAc2dHex 1832.63 NeuAc2Hex3HexNAc3dHex 1840.65NeuAcHex3HexNAc3dHex3 1841.67 NeuAc2Hex4HexNAc3 1856.64NeuAcHex4HexNAc3dHex2 1857.66 Hex5HexNAc4dHexSP 1865.60NeuAcHex4HexNAc3dHex(SP)2 1871.52 NeuAcHex5HexNAc3dHex/ 1873.66NeuGcHex4HexNAc3dHex2 Hex6HexNAc4SP 1881.65 NeuAcHex5HexNAC3(SP)21887.51 NeuAcHex6HexNAc3 1889.65 NeuAcHex3HexNAc4dHex2 1898.69Hex4HexNAc5dHexSP 1906.63 NeuAcHex6HexNAc2dHexSP/ 1912.59NeuAcHex3HexNAc4dHex(SP)2 NeuAcHex4HexNAc4dHex 1914.68NeuAc2Hex3HexNAc3dHexSP 1920.60 Hex5HexNAc5SP 1922.62NeuAcHex4HexNAc4(SP)2 1928.54 NeuAcHex5HexNAc4 1930.68 NeuGcHex5HexNAc41946.67 NeuAcHex5HexNAc3dHexSP 1953.62 NeuAcHex3HexNAc5dHex 1955.71NeuAc2Hex5HexNAc2dHex/ 1961.67/ Hex6HexNAc4(SP)2 1961.55NeuAcHex4HexNAc5 1971.71 NeuAcHex5HexNAc4Ac 1972.69NeuAcHex6HexNAc2dHex2/ 1978.69/ NeuAcHex3HexNAc4dHex2SP 1978.65NeuAc2Hex4HexNAc3dHex/ 2002.70/ Hex8HexNAc3SP 2002.62NeuAcHex4HexNAc3dHex3 2003.72 NeuAcHex5HexNAc4SP 2010.64Hex5HexNAc4dHex2SP 2011.66 NeuAc2Hex5HexNAc3/ 2018.70NeuGcNeuAcHex4HexNAc3dHex NeuAcHex5HexNAc3dHex2 2019.72NeuGcHex5HexNAc4SP 2026.63 Hex6HexNAc4dHexSP 2027.65NeuAcHex6HexNAc3dHex 2035.71 NeuAc2Hex3HexNAc4dHex/ 2043.73/Hex7HexNAc4SP 2043.65 NeuAcHex7HexNAc3 2051.71 Hex4HexNAc5dHex2SP2052.68 NeuAc2Hex4HexNAc4 2059.72 NeuAcHex4HexNAc4dHex2 2060.74Hex5HexNAc5dHexSP 2068.68 NeuAcHex4HexNAc4dHex(SP)2 2074.60NeuAcHex5HexNAc4dHex 2076.74 NeuAc2Hex4HexNAc3dHexSP 2082.66NeuGc2Hex4HexNAc4 2091.71 NeuAcHex6HexNAc4/ 2092.73 NeuGcHex5HexNAc4dHexNeuAc2Hex5HexNAc3SP/ 2098.65 NeuGcNeuAcHex4HexNAc3dHexSPNeuAcHex5HexNAc3dHex2SP/ 2099.67 NeuGcHex4HexNAc3dHex3SPNeuAc2Hex3HexNAc5 2100.75 NeuAcHex3HexNAc5dHex2/ 2101.77/NeuAc2Hex4HexNAc4Ac 2101.73 NeuAcHex6HexNAc3dHexSP 2115.67NeuAcHex4HexNAc5dHex 2117.76 Hex7HexNAc3dHex2SP/ 2132.68/NeuAc2Hex3HexNAc3dHex3 2132.76 NeuAcHex5HexNAc5 2133.76Hex8HexNAc3dHexSP/ 2148.68 NeuAc2Hex4HexNAc3dHex2 NeuAcHex8Hexnac2dHex/2156.74/ NeuAcHex5HexNAc4dHexSP 2156.69 Hex5HexNAC4dHex3SP 2157.71NeuAc2Hex5HexNAc3dHex 2164.75 NeuAcHex5HexNAc3dHex3 2165.77NeuAcHex9HexNAc2/ 2172.73/ NeuAcHex6HexNAc4SP/ 2172.69NeuGcHex5HexNAc4dHexSP NeuAcHex4Hexnac6 2174.79 NeuAc2Hex6HexNAc3/2180.75 NeuGc2Hex4HexNAc3dHex2 NeuAcHex6HexNAc3dHex2 2181.77NeuAc3Hex3HexNAc4/ 2188.76/ NeuGcHex6HexNAc4SP/ 2188.68NeuAc2NeuGcHex2HexNAc4dHex NeuAc2Hex3HexNAc4dHex2/ 2189.79/Hex7HexNAc4dHexSP 2189.70 NeuAcHex3HexNAc4dHex4 2190.81NeuGcNeuAcHex6HexNAc3/ 2196.74 NeuGc2Hex5HexNAc3dHex Hex4HexNAc5dHex3SP2198.74 NeuAc2Hex4HexNAc4dHex 2205.78 NeuAcHex4HexNAc4dHex3 2206.80NeuAc2Hex4HexNAc4(SP)2 2219.64 NeuAc2Hex5HexNAc4 2221.78NeuAcHex5HexNAc4dHex2 2222.80 Hex6HexNAc5dHexSP 2230.73NeuGcNeuAcHex5HexNAc4 2237.77 NeuAcHex6HexNAc4dHex/ 2238.79NeuGcHex5HexNAc4dHex2 NeuAc2Hex3HexNAc5dHex 2246.81NeuAcHex3HexNAc5dHex3 2247.83 NeuGc2Hex5Hexnac4 2253.76NeuAcHex7HexNAc4/ 2254.79 NeuGcHex6HexNAc4dHex NeuAc2Hex4HexNAc5 2262.80NeuAcHex4HexNAc5dHex2/ 2263.82/ NeuAc2Hex5HexNAc4Ac 2263.79NeuAcHex5HexNAc5dHex 2279.82 NeuAc2Hex4HexNAc4dHexSP 2285.74NeuAcHex4HexNAc4dHex3SP 2286.76 NeuAcHex8HexNAc3SP/ TC 2293.72/NeuAc3Hex4HexNAc3dHex 2293.80 NeuAc2Hex4HexNAc3dHex3 2294.82NeuAcHex6HexNAc5 2295.81 NeuAc2Hex5HexNAc4SP 2301.73NeuAcHex5HexNAc4dHex2SP 2302.75 NeuAc2Hex5HexNAc4Ac2 2305.80NeuAc2Hex5HexNAc3dHex2/ 2310.81 NeuGcNeuAcHex4HexNAc3dHex3NeuAcHex5HexNAc3dHex4/ 2311.83 NeuGcHex6HexNAc5 NeuAcHex6HexNAc4dHexSP2318.75 Hex6HexNAc4dHex3SP/ 2319.77 NeuGcNeuAcHex3HexNAc6NeuAcHex4HexNAc6dHex 2320.84 NeuAcHex5HexNAc5dHexAc 2321.83NeuAc2Hex6HexNAc3dHex 2326.81 NeuAcHex6HexNAc3dHex3 2327.83NeuAcHex7HexNAc4SP/ 2334.74/ NeuGcHex6HexNAc4dHexSP/ 2334.79NeuAcHex10HexNAc2 NeuAcHex5HexNAc6 2336.84 NeuAc3Hex4HexNac4 2350.82NeuAc2Hex4HexNAc4dHex2/ 2351.84/ Hex8HexNAc4dHexSP 2351.76NeuGcNeuAc2Hex4HexNAc4 2366.81 NeuAc2Hex5HexNAc4dHex 2367.83NeuAcHex5HexNAc4dHex3 2368.85 NeuAcHex5HexNAc4dHex2(SP)2 2382.71NeuAc2Hex6HexNAc4/ 2383.83 NeuGcNeuAcHex5HexNAc4dHexNeuAcHex6HexNAc4dHex2/ 2384.85 NeuGcHex5HexNAc4dHex3NeuAc3Hex5HexNAc3SP/ 2389.75/ NeuAc2Hex5HexNAc4Ac4 2389.82NeuAc2Hex5HexNAc3dHex2SP 2390.77 NeuAcHex5HexNAc3dHex4SP/ 2391.79/NeuAc3Hex3HexNAc5 2391.84 NeuAc2Hex3HexNAc5dHex2 2392.86NeuAcHex3HexNAc5dHex4 2393.89 NeuGc2Hex5HexNAc4dHex 2399.82Hex4HexNAc6dHex3SP 2401.82 NeuAc2Hex6HexNAc3dHexSP 2406.76NeuAc2Hex4HexNAc5dHex 2408.86 NeuAcHex4HexNAc5dHex3/ 2409.88/NeuAc2Hex5HexNAc4dHexAc 2409.84 NeuAc2Hex5HexNAc5 2424.85NeuAcHex5HexNAc5dHex2 2425.87 NeuAcHex8HexNAc3dHexSP/ 2439.77NeuAc3Hex4HexNAc3dHex2 NeuAcHex6HexNAc5dHex 2441.87NeuAc2Hex8HexNAc2dHex/ 2447.83/ NeuAc2Hex5HexNAc4dHexSP 2447.79NeuAcHex8HexNAc2dHex3/ 2448.85/ NeuAcHex5HexNAc4dHex3SP 2448.81NeuAcHex3HexNAc6dHex3 2450.91 NeuAc2Hex5HexNAc4dHexAc2 2451.85NeuAc2Hex5HexNAc3dHex3 2456.87 NeuAcHex7HexNAc5 2457.86NeuAcHex5HexNAc5dHex2Ac 2467.89 NeuAc2Hex6HexNAc3dHex2 2472.86NeuAcHex6HexNAc3dHex4/ 2473.88 NeuGcHex7HexNAc5 NeuAcHex5HexNAc6dHex2482.90 NeuAcHex6HexNAc5Ac 2483.88 NeuAc2Hex7HexNAc3dHex 2488.86NeuAcHex7HexNAc3dHex3 2489.88 NeuAcHex6HexNAc6/ 2498.89NeuGcHex5hexNAc6dHex NeuAc3Hex5HexNAc4 2512.87 NeuAc2Hex5HexNAc4dHex22513.89 NeuAcHex5HexNAc4dHex4 2514.91 NeuAcHex6HexNAc5dHexSP/ 2521.83/NeuAcHex9HexNAc3dHex/ 2521.87 NeuAc3Hex2HexNAc5dHex2 Hex6HexNAc5dHex3SP2522.85 NeuGcNeuAc2Hex5HexNAc4 2528.87 NeuAc2Hex6HexNAc4dHex/ 2529.89NeuGcNeuAcHex5HexNAc4dHex2 NeuAcHex6HexNAc4dHex3 2530.91NeuAc3Hex3HexNAc5dHex/ 2537.90/ NeuGcHex6HexNAc5dHexSP/ 2537.82NeuAcHex7HexNAc5SP NeuAc2Hex3HexNAc5dHex3 2538.92 NeuAcHex5HexNAc7/2539.92 NeuAcHex3HexNAc5dHex5 NeuGc2NeuAcHex5HexNAc4 2544.86NeuGc2Hex5Hexnac4dHex2/ 2545.88 NeuGcNeuAcHex6HexNAc4dHexNeuAc3Hex4HexNAc5 2553.90 NeuAc2Hex4HexNAc5dHex2 2554.92NeuAcHex4HexNAc5dHex4 2555.94 NeuGc3Hex5HexNAc4 2560.86NeuAc2Hex5HexNAc5dHex 2570.91 NeuAcHex5HexNAc5dHex3 2571.93NeuAc2Hex6HexNAc5 2586.91 NeuAcHex6HexNAc5dHex2 2587.93Hex7HexNAc6dHexSP 2595.86 NeuGcNeuAcHex6HexNAc5 2602.90NeuAcHex7HexNAc5dHex/ 2603.92/ NeuGcHex6HexNAc5dHex2 603.92NeuGc2Hex6HexNac5 2618.90 NeuAcHex8HexNAc5/ 2619.92 NeuGcHex7HexNAc5dHexNeuAc2Hex5HexNAc6 2627.93 NeuAcHex5HexNAc6dHex2 2628.95NeuGcHex8HexNAc5/ 2635.91/ NeuAcHex4HexNAc5dHex4SP 2635.89NeuAcHex6HexNAc6dHex 2644.95 NeuAc2Hex5HexNAc5dHexSP 2650.87NeuAc2Hex5HexNAc4dHex3 2659.95 NeuAcHex7HexNAc6 2660.94NeuGcNeuAc2Hex5HexNAc4dHex 2674.92 NeuAc3Hex6HexNAc4NeuGcHex6HexNAc5dHexSP/ 2683.88 NeuAcHex7HexNAc5dHexSPNeuAcHex5HexNAc7dHex 2685.98 NeuAc2Hex7HexNAc4dHex 2691.94NeuAcHex7HexNAc4dHex3 2692.96 NeuAc2Hex4HexNAc5dHex2(SP)2 2714.83NeuAcHex4HexNAc5dHex4(SP)2/ 2715.85/ NeuAc3Hex5HexNAc5 2715.95NeuAc2Hex5HexNAc5dHex2 2716.97 NeuAcHex5HexNAc5dHex4 2717.99NeuAc2Hex6HexNAc5dHex 2732.97 NeuAcHex6HexNAc5dHex3 2733.99NeuAcHex6HexNAc5dHex2(SP)2 2747.84 NeuGcNeuAcHex6HexNAc5dHex 2748.96NeuAc3Hex4HexNAc6 2756.98 NeuAc2Hex4HexNAc6dHex2 2758.00NeuAcHex4HexNAc6dHex4 2759.02 NeuAc3Hex6HexNAc3dHex2 2763.96NeuAc2Hex6HexNAc3dHex4/ 2764.98/ NeuGc2Hex6HexNAc5dHex/ 2764.96NeuGcHex7HexNAc5 NeuAcHex8HexNAc5dHex 2765.98 NeuAc2Hex5HexNAc6dHex2773.99 NeuAcHex5HexNAc6dHex3 2775.01 NeuGc2Hex7HexNAc5 2780.95NeuGcHex8HexNAc5dHex/ 2781.97 NeuAcHex9HexNac5 NeuAc2Hex6HexNAc6 2789.99NeuAc4Hex6HexNAc6dHex2 2791.01 NeuAc4Hex5HexNAc4 2803.97NeuAc3Hex5HexNAc4dHex2/ 2804.99/ NeuAcHex6HexNAc6dHex(SP)2 2804.86Hex6HexNAc6dHex3SP2 2805.88 NeuAc2Hex5HexNAc4dHex4 2806.01NeuAcHex7Hexnac6dHex 2807.00 NeuAc2Hex6HexNAc5dHexSP 2812.92NeuAcHex6HexNAc5dHex3SP 2813.94 NeuGcNeuAc3Hex5HexNAc4 2819.96NeuAc3Hex6HexNAc4dHex/ 2820.98 NeuGcNeuAc2Hex5HexNAc4dHex2NeuAc2Hex6HexNAc4dHex3 2822.00 NeuAcHex8HexNAc6 2823.00NeuGc2NeuAc2Hex5HexNAc4 2835.96 NeuGc2NeuAcHex5HexNAc4dHex2 2836.98NeuAc3Hex6HexNAc5 2878.00 NeuAc2Hex6HexNAc5dHex2 2879.02NeuAcHex6HexNAc5dHex4 288.04 NeuAcHex7HexNAc6dHexSP/ 2886.96/NeuAcHex10HexNAc4dHex 2887.00 NeuGcNeuAc2Hex6HexNAc5 2894.00NeuAc2Hex7HexNAc5dHex/ 2895.02 NeuGcNeuAcHex6HexNAc5dHex2NeuAc3Hex6HexNAc4dHexSP/ 2900.94 NeuGcNeuAc2Hex5HexNAc4dHex2SPNeuGc2NeuAcHex6HexNAc5 2909.99 NeuGc2Hex6HexNAc5dHex2 2911.01NeuAc3Hex5HexNAc6 2919.03 NeuAc2Hex5HexNAc6dHex2 2920.05NeuAcHex5HexNAc6dHex4 2921.07 NeuGc3Hex6HexNAc5 2925.99NeuGcNeuAc2Hex5HexNAc6 2935.02 NeuAc2Hex6HexNAc6dHex/ 2936.04NeuGcNeuAcHex5HexNAc6dHex2 NeuAcHex6HexNAc6dHex3 2937.07NeuGc2NeuAcHex5HexNAc6/ 2951.02/ NeuAc3Hex5HexNAc4dHex3 2951.04NeuAc2Hex7HexNAc6 2952.04 NeuAcHex7HexNAc6dHex2 2953.06NeuAc2Hex6HexNAc5dHex2SP 2958.98 NeuAcHex6HexNAc5dHex4SP 2960.00NeuAc2Hex4HexNAc7dHex2 2961.08 NeuAcHex4HexNAc7dHex4 2962.10NeuAcHex6HexNAc7dHex2 2994.09 NeuAcHex7HexNAc7dHex 3010.08NeuAc3Hex6HexNAc5dHex 3024.06 NeuAc2Hex6HexNAc5dHex3 3025.08NeuAcHex8HexNAc7 3026.08 NeuAc3Hex5HexNAc6dHex 3065.09NeuAc2Hex5HexNAc6dHex3 3066.11 NeuAcHex7HexNAc8 3067.10NeuAc3Hex6HexNAc6 3081.08 NeuAc2Hex6HexNAc6dHex2 3082.10NeuAc2Hex7HexNAc6dHex 3098.10 NeuAcHex7HexNAc6dHex3 3099.12NeuAc3Hex6HexNAc5dHexSP 3104.02 NeuAc2Hex6HexNAc5dHex3SP 3105.04NeuAcHex8HexNAc7SP/ 3106.03/ NeuAc3Hex4HexNAc7dHex 3106.11Hex8HexNAc7dHex2SP/ 3107.05/ NeuAc2Hex4HexNAc7dHex3 3107.13NeuAcHex7HexNAc7dHex2 3156.14 NeuAc3Hex6HexNAc5dHex2 3170.12NeuAc2Hex6HexNAc5dHex4 3171.14 NeuAcHex8HexNAc7dHex 3172.13NeuAc2Hex7HexNAc6dHexSP 3178.05 NeuAc3Hex6HexNAc6dHex 3227.14NeuAc2Hex6HexNAc6dHex3 3228.16 NeuAcHex8HexNAc8 3229.16NeuAc3Hex7HexNAc6 3243.13 NeuAc2Hex7HexNAc6dHex2 3244.16NeuAcHex7HexNAc6dHex4 3245.18 NeuAc2Hex8HexNAc6dHex/ 3260.15NeuGcNeuAcHex7HexNAc6dHex2 NeuAcHex8HexNAc6dHex3/ 3261.17NeuGcHex7HexNAc6dHex4 NeuAc3Hex7HexNAc5dHexSP/ 3266.07NeuGcNeuAc2Hex6HexNAc5dHex2SP NeuAc3Hex5HexNAc7dHex/ 3268.17/NeuGcHex8HexNAc7dHexSP 3268.09 NeuAc2Hex5HexNAc7dHex3 3269.19NeuAcHex7HexNAc9 3270.18 NeuGc2Hex7HexNAc6dHex2 3276.15NeuAc4Hex4HexNAc5dHex2(SP)2 3297.02 NeuAc3Hex4HexNAc5dHex4(SP)2 3298.04NeuAc2Hex7HexNAc7dHex 3301.18 NeuAcHex7HexNAc7dHex3 3302.20NeuAc3Hex6HexNAc5dHex3 3316.18 NeuAc2Hex8HexNAc7 3317.17NeuAcHex8HexNAc7dHex2 3318.19 NeuAc3Hex7HexNAc6dHex 3389.19NeuAc2Hex7HexNAc6dHex3 3390.21 NeuAcHex7HexNAc6dHex5/ 3391.23NeuAcHex9HexNAc8 NeuAc3Hex5HexNAc7dHex2 3414.22 NeuAc2Hex5HexNAc7dHex43415.24 NeuAcHex7HexNAc9dHex 3416.24 NeuAc3Hex6HexNAc7dHex 3430.22NeuAc2Hex6HexNAc7dHex3 3431.24 NeuAcHex8HexNAc9 3432.24NeuAc2Hex8Hexnac7dHex 3463.23 NeuAcHex8HexNAc7dHex3 3464.25NeuAc3Hex7HexNAc6dHexSP 3469.15 NeuAc2Hex7HexNAc6dHex3SP 3470.17NeuAc3Hex5HexNAc8dHex 3471.25 NeuAc2Hex5HexNAc8dHex3 3472.27NeuAcHex7HexNAc10 3473.26 NeuAc4Hex7HexNAc6 3534.23NeuAc3Hex7HexNAc6dHex2 3535.25 NeuAc2Hex7HexNAc6dHex4 3536.27NeuAcHex9HexNAc8dHex 3537.27 NeuAc4Hex5HexNAc7dHex 3559.26NeuAc3Hex5HexNAc7dHex3 3560.28 NeuAc2Hex7HexNAc9 3561.28NeuAcHex7HexNAc9dHex2 3562.30 NeuAc3Hex7HexNac7dHex 3592.27NeuAc2Hex7HexNAc7dHex3 3593.29 NeuAcHex9HexNAc9 3594.29NeuAc3Hex8HexNAc7 3608.27 NeuAc2Hex8HexNac7dHex2 3609.29NeuAcHex8HexNac7dHex4 3610.31 NeuAc3Hex5HexNAc8dHex2 3617.30NeuAc2Hex5HexNAc8dHex4 3618.32 NeuAcHex7HexNAc10dHex 3619.32NeuAc3Hex6HexNAc8dHex 3633.30 NeuAc4Hex7HexNAc6dHex 3680.29NeuAc3Hex7HexNAc6dHex3 3681.31 NeuAc2Hex9HexNAc8 3682.30NeuAcHex9HexNAc8dHex2 3683.32 NeuAc4Hex6HexNAc7dHex 3721.31NeuAc3Hex6HexNAc7dHex3 3722.34 NeuAc2Hex8HexNAc9 3723.33NeuAcHex8HexNAc9dHex2 3724.35 NeuAc3Hex7HexNac7dHex2 3738.33NeuAc2Hex7HexNAc7dHex4 3739.35 NeuAcHex9HexNAc9dHex 3740.35NeuAc3Hex8HexNAc7dHex 3754.33 NeuAc2Hex8HexNAc7dHex3 3755.35NeuAcHex10HexNAc9/ 3756.34 NeuAcHex8HexNAc7dHex5 NeuAc4Hex6HexNAc83778.34 NeuAc3Hex6HexNAc8dHex2 3779.36 NeuAc2Hex6HexNAc8dHex4 3780.38NeuAcHex8HexNAc10dHex 3781.37 NeuAc4Hex7HexNAc6dHex2 3826.35NeuAc3Hex7Hexnac6dHex4 3827.37 NeuAc2Hex9HexNAc8dHex 3828.36NeuAcHex9HexNAc8dHex3 3829.38 NeuAc4Hex8HexNAc7 3899.36NeuAc3Hex8HexNAc7dHex2 3900.38 NeuAc2Hex8HexNAc7dHex4 3901.40NeuAcHex10HexNAc9dHex 3902.40 NeuAc4Hex6HexNAc8dHex 3924.39NeuAc3Hex6HexNAc8dHex3 3925.41 NeuAc2Hex8HexNAc10 3926.41NeuAcHex8HexNAc10dHex2 3927.43 NeuAc3Hex9HexNAc8 3973.40NeuAc2Hex9HexNAc8dHex2 3974.42 NeuAcHex9HexNAc8dHex4 3975.44NeuAc4Hex8HexNAc7dHex 4045.42 NeuAc3Hex8HexNAc7dHex3 4046.44NeuAc2Hex10HexNAc9/ 4047.44 NeuAc2Hex8HexNAc7dHex5NeuAcHex10HexNAc9dHex2 4048.46 NeuAc3Hex9HexNAc8dHex 4119.46NeuAc2Hex9HexNAc8dHex3 4120.48 NeuAcHex11HexNAc10/ 4121.47NeuAcHex9HexNAc8dHex5 NeuAc2Hex10HexNAc9dHex2 4339.55NeuAcHex10HexNAc9dHex4 4340.57 NeuAc2Hex10HexNAc9dHex3 4485.61

TABLE 3 Neutral N-glycan profiles of cord blood mononuclear cellpopulations and peripheral blood mononuclear cells. Proposedmonosaccharide composition calc. m/z CD34+ CD34− CD133+ CD133− LIN− LIN+CB MNC PB MNC HexHexNAc2 609.21 2.14 0.15 0.22 0.22 HexHexNAc2dHex755.27 0.40 0.83 0.37 0.95 0.28 0.60 Hex2HexNAc2 771.26 2.37 4.27 2.293.30 1.72 2.62 3.34 4.19 Hex2HexNAc2dHex 917.32 9.10 13.32 5.97 11.616.75 8.24 7.54 8.41 Hex3HexNAc2 933.31 8.20 5.84 4.85 5.02 3.94 3.714.53 5.67 Hex2HexNAc3 974.34 0.02 Hex3HexNAc2dHex 1079.38 7.32 6.70 4.907.02 4.41 5.51 5.36 6.45 Hex4HexNAc2 1095.37 5.28 4.21 4.79 4.13 3.923.38 4.36 4.39 Hex2HexNAc3dHex 1120.40 0.14 0.17 0.09 0.04 Hex3HexNAc31136.40 1.25 0.75 0.24 0.58 3.01 0.50 0.41 0.43 Hex3HexNAc2dHex2 1225.430.10 Hex4HexNAc2dHex 1241.43 0.43 0.36 0.27 0.63 0.40 0.57 0.51 0.53Hex5HexNAc2 1257.42 16.90 18.53 20.40 13.88 18.05 14.92 15.80 15.32Hex3HexNAc3dHex 1282.45 1.15 1.74 1.14 1.77 1.55 1.43 0.96 0.94Hex4HexNAc3 1298.45 0.35 0.60 0.20 0.43 1.53 0.52 0.43 0.49HexHexNAc4dHex2 1307.49 0.40 Hex3HexNAc4 1339.48 0.54 1.18 0.31 0.170.19 Hex5HexNAc2dHex 1403.48 0.19 0.45 0.57 0.57 0.39 0.55 0.53 0.53Hex6HexNAc2 1419.48 11.87 13.37 15.93 15.94 11.33 16.14 17.98 16.44Hex3HexNAc3dHex2 1428.51 0.48 0.43 0.23 0.09 0.17 Hex4HexNAc3dHex1444.51 0.65 0.84 0.56 0.54 0.73 0.40 0.36 Hex5HexNAc3 1460.50 0.28 0.330.33 0.45 0.83 0.56 0.56 0.47 Hex3HexNAc4dHex 1485.53 1.55 1.22 2.882.07 4.90 3.38 0.91 1.02 Hex4HexNAc4 1501.53 0.18 0.13 0.20 0.82 0.080.01 0.09 Hex3HexNAc5 1542.56 0.28 0.06 0.38 0.03 0.02 0.01Hex6HexNAc2dHex 1565.53 0.11 0.09 0.08 0.11 0.15 0.15 Hex7HexNAc21581.53 8.68 8.04 9.78 10.16 9.58 11.24 11.50 11.28 Hex4HexNAc3dHex21590.57 0.72 1.01 0.46 0.25 0.37 0.16 Hex5HexNAc3dHex 1606.56 0.10 0.080.10 0.22 0.31 0.31 0.20 0.14 Hex6HexNAc3 1622.56 0.37 0.34 0.39 0.640.80 0.78 0.72 0.57 Hex4HexNAc4dHex 1647.59 0.37 0.35 0.52 0.22 0.630.82 0.08 0.13 Hex5HexNAc4 1663.58 0.39 0.84 0.64 0.99 0.93 0.51 0.70Hex3HexNAc5dHex 1688.61 0.26 0.43 0.54 0.59 0.79 0.65 0.47 0.49Hex4HexNAc5 1704.61 0.09 0.14 0.03 Hex7HexNAc2dHex 1727.59 0.03Hex8HexNAc2 1743.58 8.51 5.69 10.36 7.19 9.04 8.53 9.18 9.31Hex5HexNAc3dHex2 1752.62 0.05 0.06 0.06 Hex6HexNAc3dHex 1768.61 0.050.02 0.13 0.09 0.10 Hex7HexNAc3 1784.61 0.06 0.05 0.03 0.05Hex4HexNAc4dHex2 1793.64 0.05 0.18 0.15 0.09 0.08 Hex5HexNAc4dHex1809.64 0.59 0.64 0.41 0.36 0.68 0.42 0.22 0.24 Hex6HexNAc4 1825.63 0.070.13 0.26 0.06 Hex5HexNAc5 1866.66 0.05 0.09 0.08 0.23 0.03Hex3HexNAc6dHex 1891.69 0.23 0.16 0.14 0.06 0.15 Hex9HexNAc2 1905.6310.07 6.75 9.80 7.17 10.11 9.49 9.55 9.09 Hex5HexNAc4dHex2 1955.70 0.320.33 0.17 0.08 0.15 0.10 Hex6HexNAc4dHex 1971.69 0.03 0.06 0.00Hex7HexNAc4 1987.69 0.02 0.07 0.01 Hex5HexNAc5dHex 2012.72 0.04Hex6HexNAc5 2028.71 0.10 0.14 0.10 0.08 Hex10HexNAc2 2067.69 0.27 0.530.69 0.67 0.63 0.87 1.14 1.14 Hex5HexNAc4dHex3 2101.76 0.22 0.37 0.030.23 0.13 0.13 0.08 Hex6HexNAc4dHex2 2117.75 0.06 Hex8HexNAc4 2149.740.05 Hex6HexNAc5dHex 2174.77 0.08 0.04 0.05 0.12 0.02 Hex4HexNAc6dHex22199.80 0.01 Hex5HexNAc6dHex 2215.80 0.01 Hex11HexNAc2 2229.74 0.05 0.020.15 0.10 Hex6HexNAc6 2231.79 0.01 Hex6HexNAc5dHex2 2320.83 0.02Hex12HexNAc2 2391.79 0.02 0.10 0.04 0.12 0.05 Hex7HexNAc6 2393.85 0.02Hex6HexNAc7 2434.87 0.25 Hex6HexNAc5dHex3 2466.89 0.01 Hex7HexNAc6dHex2539.90 0.01

TABLE 4 Sialylated N-glycan profiles of cord blood mononuclear cellpopulations and peripheral blood mononuclear cells. Proposed CDmonosaccharide composition calc. m/z CD 34+ 34− MNC NeuAcHex2HexNAc835.28 0.15 NeuAcHex2HexNAc2 1038.36 0.12 Hex4HexNAc2SP 1151.33 0.25NeuAcHex3HexNAc2 1200.42 0.54 1.06 0.47 NeuAc2HexHexNAc2dHex 1313.460.22 NeuAc2Hex2HexNAc2 1329.46 0.60 NeuAcHex4HexNAc2 1362.47 0.54NeuAcHex3HexNAc3 1403.49 0.62 0.47 0.38 NeuAc2Hex2HexNAcdHex 1475.520.59 0.67 NeuAc2Hex3HexNAc2dHex 1491.51 0.22 NeuAcHex3HexNAc3dHex1549.55 1.72 1.01 1.61 NeuAc2Hex2Hexnac2dHexSP 1555.47 0.35NeuAcHex3HexNAc3SP2 1563.41 0.63 3.41 NeuAcHex4HexNAc3 1565.55 1.99 0.422.36 NeuAc2Hex3HexNAc2dHex 1637.57 0.47 0.55 NeuAc2Hex2HexNAc3dHex1678.60 0.38 0.59 NeuAcHex3HexNAc3dHexSP2 1709.47 0.08NeuAcHex4HexNAc3dHex 1711.61 6.44 1.45 7.21 NeuAcHex5HexNAc3 1727.601.23 0.53 1.83 NeuAc2Hex2HexNAc3dHexSP 1758.55 0.39 NeuAcHex4HexNAc41768.57 1.55 0.64 1.39 NeuAcHex4HexNAc3dHexSP 1791.56 0.09NeuAc2Hex4HexNAc2dHex 1799.62 0.12 NeuAc2Hex5HexNAc2/ 1815.62/ 0.47 0.18NeuAc2Hex2HexNAc4SP 1815.57 NeuAc2Hex4HexNAc3 1856.64 0.28NeuAcHex4HexNAc3dHex2 1857.66 0.04 Hex5HexNAc4dHexSP 1865.60 0.13NeuAcHex5HexNAc3dHex 1873.66 1.50 0.27 1.80 NeuAcHex6HexNAc3 1889.651.21 0.26 2.67 NeuAcHex6HexNAc2dHexSP/ 1912.59/ 0.60 0.26NeuAcHex3HexNAc4dHexSP2 1912.55 NeuAcHex4HexNAc4dHex 1914.68 2.80 1.152.64 NeuAc2Hex3HexNAc3dHexSP 1920.60 0.22 NeuAcHex4HexNAc4SP2 1928.540.26 NeuAcHex5HexNAc4 1930.68 10.25 2.87 10.12 NeuGcHex5HexNAc4 1946.670.10 NeuAc2Hex4HexNAc3dHex/ 2002.70/ 0.65 Hex8HexNAc3SP 2002.62NeuAc2Hex5HexNAc3 2018.70 0.57 1.27 NeuAcHex5HexNAc3dHex2 2019.72 0.170.09 NeuAcHex6HexNAc3dHex 2035.71 0.78 0.71 NeuAcHex7HexNAc3 2051.710.15 NeuAc2Hex4HexNAc4 2059.72 0.25 NeuAcHex4HexNAc4dHex2 2060.74 0.20NeuAcHex4HexNAc4dHexSP2 2074.60 0.78 0.13 NeuAcHex5HexNAc4dHex 2076.7410.89 4.35 14.12 NeuAcHex6HexNAc4 2092.73 0.17 NeuAc2Hex5HexNAc3SP/2098.65 0.24 NeuGcNeuAcHex4HexNAc3dHexSP NeuAcHex5HexNAc3dHex2SP/2099.67 0.07 NeuGcHex4HexNAc3dHex3SP NeuAcHex4HexNAc5dHex 2117.76 0.570.13 0.52 NeuAcHex5HexNAc5 2133.76 0.55 1.07 NeuAcHex8HexNAc2dHex/2156.74/ 0.42 NeuAcHex5HexNAc4dHexSP 2156.69 NeuAc2Hex4HexNAc4dHex2205.78 0.26 NeuAc2Hex4HexNAc4SP2 2219.64 0.45 0.57 NeuAc2Hex5HexNAc42221.78 13.41 10.38 9.12 NeuAcHex5HexNAc4dHex2 2222.80 3.80 2.21 3.28Hex6HexNAc5dHexSP 2230.73 0.09 NeuGcNeuAcHex5HexNAc4 2237.77 0.61 0.69NeuAcHex6HexNAc4dHex/ 2238.79 0.20 0.13 0.29 NeuGcHex5HexNAc4dHex2NeuGc2Hex5HexNAc4 2253.76 0.44 NeuAcHex7HexNAc4/ 2254.79 0.05NeuGcHex6HexNAc4dHex NeuAcHex5HexNAc5dHex 2279.82 0.91 0.72 2.06NeuAcHex8HexNAc3SP 2293.72 0.20 NeuAcHex6HexNAc5 2295.81 0.56 0.30 1.63NeuAc2Hex5HexNAc4SP 2301.73 0.12 NeuAc2Hex4HexNAc4dHexSP2 2365.69 1.111.70 NeuAc2Hex5HexNAc4dHex 2367.83 12.90 17.84 11.02NeuAcHex5HexNAc4dHex3 2368.85 3.38 2.05 2.03 NeuAcHex5HexNAc4dHex2SP22382.71 0.28 NeuAc2Hex6HexNAc4 2383.83 0.21 NeuAcHex6HexNAc4dHex22384.85 0.21 NeuAc2Hex5HexNAc3dHex2SP 2390.77 0.68 0.58 2.18NeuAcHex5HexNAc5 2424.85 0.58 0.39 0.29 NeuAcHex5HexNAc5dHex2 2425.870.12 0.46 NeuAcHex8HexNAc3dHexSP 2439.77 0.21 NeuAcHex6HexNAc5dHex2441.87 1.60 1.30 4.40 NeuAc2Hex8HexNAc2dHex/ 2447.83/ 0.60 2.25NeuAc2Hex5HexNAc4dHexSP 2447.79 NeuAcHex8HexNAc2dHex3/ 2448.85/ 0.18NeuAcHex5HexNAc4dHex3SP 2448.81 NeuAcHex6HexNAc3dHex4/ 2473.88 0.21NeuGcHex7HexNAc5 NeuAcHex7HexNAc3dHex3 2489.88 0.77NeuAc2Hex5HexNAc4dHex2 2513.89 0.50 0.61 NeuAcHex6HexNAc5dHexSP/2521.83/ 0.08 NeuAcHex9HexNAc3dHex/ 2521.87 NeuAc3Hex2HexNAc5dHex2NeuGcNeuAc2Hex5HexNAc4 2528.87 0.34 NeuAc2Hex6HexNAc4dHex/ 2529.89/ 0.05NeuGcNeuAcHex5HexNAc4dHex2 2529.89 NeuGc2NeuAcHex5HexNAc4 2544.86 0.13NeuAc2Hex5HexNAc5dHex 2570.91 0.81 1.78 0.99 NeuAcHex5HexNAc5dHex32571.93 0.33 0.25 0.19 NeuAc2Hex6HexNAc5 2586.91 0.97 0.52NeuAcHex6HexNAc5dHex2 2587.93 1.00 0.28 0.76 NeuAcHex7HexNAc5dHex/2603.92 0.09 NeuGcHex6HexNAc5dHex2 NeuAcHex8HexNAc5/ 2619.92 0.38 0.31NeuGcHex7HexNAc5dHex NeuGcHex8HexNAc5/ 2635.91/ 0.65 0.13NeuAcHex4HexNAc5dHex4SP 2635.89 NeuAcHex6HexNAc6dHex 2644.95 0.64NeuAc2Hex5HexNAc5dHexSP 2650.87 0.14 NeuAcHex7HexNAc6 2660.94 0.42NeugcNeuAc2Hex5HexNAc4dHex 2674.92 0.14 NeuAc2Hex4HexNAc5dHex2SP22714.83 0.24 NeuAc2Hex5HexNAc5dHex2 2716.97 0.21 NeuAc2Hex6HexNAc5dHex2732.97 1.70 4.43 2.88 NeuAcHex6HexNAc5dHex3 2733.99 0.62 1.08 1.66NeuAcHex6HexNAc5dHex2SP2 2747.84 0.21 NeuAcHex6HexNAc6dHexSP2/ 2804.86/0.18 NeuAc3Hex5HexNAc4dHex2 2804.99 NeuAcHex7HexNAc6dHex 2807.00 0.451.54 NeuAc2Hex6HexNAc5dHexSP 2812.92 0.75 NeuAc3Hex6HexNAc5 2878.00 0.970.17 NeuAc2Hex6HexNAc5dHex2 2879.02 0.72 0.41 0.46 NeuAcHex6HexNAc5dHex42880.04 0.15 0.35 NeuAc3Hex6HexNAc4dHexSP 2900.94 0.18NeuAc2Hex6HexNAc6dHex 2936.04 0.32 NeuAcHex6HexNAc6dHex3 2937.07 0.090.25 NeuAcHex7HexNAc6dHex2 2953.06 0.28 NeuAc2Hex6HexNAc5dHex2SP 2958.980.20 NeuAc3Hex6HexNAc5dHex 3024.06 1.37 7.52 0.98 NeuAc2Hex6HexNAc5dHex33025.09 0.39 1.16 0.65 NeuAcHex8HexNAc7 3026.08 0.17NeuAc2Hex7HexNAc6dHex 3098.10 0.52 0.85 0.47 NeuAcHex7HexNAc6dHex33099.12 0.44 0.24 NeuAc3Hex6HexNAc5dHexSP 3104.02 0.45 0.72NeuAc2Hex6HexNAc5dHex3SP 3105.04 0.47 NeuAc3Hex6HexNAc5dHex2 3170.120.17 NeuAc2Hex6HexNAc5dHex4 3171.14 0.02 NeuAcHex8HexNAc7dHex 3172.130.12 0.11 NeuAc2Hex7Hexnac6dHexSP 3178.05 0.10 NeuAc3Hex6HexNAc6dHex3227.14 0.33 NeuAc2Hex7HexNAc6dHex2 3244.16 0.20 NeuAcHex7HexNAc6dHex43245.18 0.19 NeuAc3Hex7Hexnac5dHexSP 3266.07 0.10 NeuGc2Hex7HexNAc6dHex23276.15 0.14 NeuAc3Hex7HexNAc6dHex 3389.19 0.13 0.74NeuAc2Hex7HexNAc6dHex3 3390.21 0.37 NeuAc2Hex8HexNAc7dHex 3463.23 0.15NeuAcHex8HexNAc7dHex3 3464.25 0.19 NeuAc3Hex7Hexnac6dHexSP 3469.15 0.04NeuAc2Hex7Hexnac6dHex3SP 3470.17 0.08 NeuAc3Hex7HexNAc6dHex2 3535.250.15 NeuAc2Hex7HexNAc6dHex4 3536.27 0.08 NeuAc4Hex7HexNAc6dHex 3680.290.40 NeuAc3Hex7HexNAc6dHex3 3681.31 0.25 NeuAc3Hex8HexNAc7dHex 3754.330.22 NeuAc2Hex8HexNAc7dHex3 3755.35 0.05

TABLE 5 Neutral N-glycan grouping of cord blood cell populations, cordblood mononuclear cells (CB MNC), and peripheral blood mononuclear cells(PB MNC). Neutral N-glycan Grouping: CD CD CD CB PB Composition GlycanGrouping 34+ CD 34− 133+ 133− LIN− LIN+ MNC MNC General N-glycangrouping: Hex₅₋₁₂HexNAc₂ high-mannose 56.3 52.9 67.0 55.1 58.9 61.2 65.462.7 Hex₁₋₄HexNAc₂dHex₀₋₁ low-mannose 33.1 35.5 25.6 32.8 21.1 24.5 26.529.6 n_(HexNAc) = 3 and n_(Hex) ≧ 2 hybrid/monoant. 5.5 6.4 2.4 5.6 8.65.5 4.3 3.7 n_(HexNAc) ≧ 4 and n_(Hex) ≧ 2 complex 4.3 4.8 4.5 5.9 11.08.0 3.1 3.3 Other types — 0.8 0.4 0.6 0.7 0.5 0.7 0.7 0.7Complex/hybrid/monoantennary N-glycan grouping: n_(dHex) ≧ 1 fucosylated67.8 70.6 81.2 66.4 49.0 66.8 58.8 56.4 n_(dHex) ≧ 2 α2/3/4-linked Fuc18.8 21.3 0.5 11.5 0 5.4 12.2 4.9 n_(HexNAc) > n_(Hex) ≧ 2 terminalHexNAc 21.3 18.3 50.8 32.1 38.7 34.2 22.7 26.9 n_(HexNAc) = n_(Hex) ≧ 5bisecting GlcNAc 0 0 0.8 0.8 0.4 2.0 0.4 0 Complex N-glycan grouping:n_(HexNAc) ≧ 5 and n_(Hex) ≧ 6 large N-glycans 1.8 6.0 0 2.5 0 4.0 3.82.4

TABLE 6 Sialylated N-glycan grouping of cord blood cell populations,cord blood mononuclear cells (CB MNC), and peripheral blood mononuclearcells (PB MNC). Sialylated N-glycan Grouping: CD CD CB CompositionGlycan Grouping 133+ 133− MNC General N-glycan grouping: n_(HexNAc) = 3and n_(Hex) ≧ 5 hybrid 5.7 3.2 7.7 n_(HexNAc) = 3 and n_(Hex) = 3 or 4monoantennary 12.1 7.5 11.6 n_(HexNAc) ≧ 4 and n_(Hex) ≧ 3 complex 76.582.6 75.8 Other types — 5.8 6.8 5.0 Complex/hybrid/monoantennaryN-glycan grouping: n_(dHex) ≧ 1 fucosylated 62.3 70.0 67.7 n_(dHex) ≧ 2α2/3/4-linked Fuc 13.3 14.9 13.3 n_(HexNAc) > n_(Hex) ≧ 3 terminalHexNAc 0.6 0.1 0.6 n_(HexNAc) = n_(Hex) ≧ 5 bisecting GlcNAc 3.4 4.9 6.3Complex N-glycan grouping: n_(HexNAc) ≧ 5 and n_(Hex) ≧ 6 largeN-glycans 13.6 34.2 24.1 Sialylation degree SD_(HexNAc) = 75 78 72n_(NeuAc/Gc):(n_(HexNAc) − 2)

TABLE 7 MALDI-TOF mass spectrometric analysis ofendoglycoceramidase-released cord blood mononuclear cell glycolipidglycans. Proposed composition calc. m/z exp. m/z A. Neutraloligosaccharides detected from glycolipids of cord blood mononuclearcells. Five major peaks are bolded. Hex2HexNAc 568.18 568.09 Hex3HexNAc730.24 730.18 Hex3HexNAcdHex 876.30 876.27 Hex4HexNAc 892.29 892.27Hex3HexNAc2 933.31 933.30 Hex5HexNAc 1054.34 1054.33 Hex4HexNAc2 1095.371095.36 Hex4HexNAc2dHex 1241.43 1241.42 Hex4HexNAc2dHex2 1387.49 1387.48Hex6HexNAc2 1419.48 1419.47 Hex5HexNAc3 1460.50 1460.49 Hex5HexNAc4dHex1606.56 1606.55 Hex5HexNac3dHex2 1752.62 752.60 Hex6HexNAc4dHex2 2117.752117.71 Hex6HexNAc4dHex3 2263.81 2263.76 B. Acidic oligosaccharidesdetected from glycolipids of cord blood mononuclear cells. Five majorpeaks are bolded. NeuAcHexHexNAc 673.23 673.95 NeuAcHex2HexNAc 835.28835.31 NeuAcHex3HexNAc 997.34 997.52 NeuAcHex3HexNAc2 1200.42 1200.62NeuAcHex4HexNAc2 1362.47 1362.80 NeuAcHex4HexNAc2dHex 1508.53 1508.89NeuAcHex2HexNAc3dHex2 1533.56 1533.66 NeuAc2Hex2HexNAc2dHexSP 1555.471555.68 NeuAcHex5HexNAc3 1727.60 1728.01 NeuAcHex5HexNAc3dHex 1873.661874.07 NeuAc2Hex3HexNAc3dHexSP 1920.60 1920.87 NeuAcHex3HexNAc5dHex32247.83 2247.99

TABLE 8 Exoglycosidase profiling of cord blood CD34+ and CD34− cellneutral N-glycan fraction. α-Man, β1,4-Gal, β1,3-Gal, and β-GlcNAc referto specific exoglycosidase enzymes as described in the text. α-Manβ1,4-Gal β1,3-Gal β-GlcNAc Proposed composition m/z CD 34+ CD 34− CD 34+CD 34− CD 34+ CD 34− CD 34+ CD 34− Hex2HexNAc 568 −− +++ +++ +++ +++HexHexNAc2 609 +++ +++ +++ +++ Hex3HexNAc 730 −−− −− − HexHexNAc2dHex755 +++ ++ − − − −− Hex2HexNAc2 771 ++ −− −− −− −− −− −− Hex4HexNAc 892−−− −−− − − Hex2HexNAc2dHex 917 −− −− −− −− −− −− Hex3HexNAc2 933 −−− −−− −− −− −− HexHexNAc3dHex 958 +++ Hex2HexNAc3 974 +++ +++ Hex5HexNAc1054 −−− −− + + − Hex3HexNAc2dHex 1079 −− −− −− − −− + Hex4HexNAc2 1095−−− −−− Hex2HexNAc3dHex 1120 + + Hex3HexNAc3 1136 −−− − −−− Hex6HexNAc1216 −−− −− − − − Hex4HexNAc2dHex 1241 −−− − − − − Hex5HexNAc2 1257 −−−−− + + + + Hex3HexNAc3dHex 1282 −−− + − − −− Hex4HexNAc3 1298 −−− −−− −Hex2HexNAc4dHex 1323 +++ Hex3HexNAc4 1339 +++ +++ Hex7HexNAc 1378−−− + + Hex5HexNAc2dHex 1403 −−− +++ Hex6HexNAc2 1419 −−− −− ++ ++ ++ ++++ Hex3HexNAc3dHex2 1428 −−− ++ +++ +++ Hex4HexNAc3dHex 1444 −−− − −−−− + Hex5HexNAc3 1460 −−− − +++ +++ −−− Hex3HexNAc4dHex 1485 − + −−−Hex4HexNAc4 1501 −−− −−− −−− −−− Hex8HexNAc 1540 −−− −−− −−− +++ −−− +++−−− Hex3HexNAc5 1542 +++ +++ +++ Hex6HexNAc2dHex 1565 +++ Hex7HexNAc21581 −−− −− ++ ++ ++ ++ Hex4HexNAc3dHex2 1590 −−− −−− − − +Hex5HexNAc3dHex 1606 −−− −−− +++ +++ +++ Hex6HexNAc3 1622 −−− −−− −−−−−− −−− Hex4HexNAc4dHex 1647 −−− − −−− Hex5HexNAc4 1663 −−− −−− −−− −−−−− −−− Hex3HexNAc5dHex 1688 +++ +++ Hex9HexNAc 1702 −−− −−− +++ +++ +++Hex4HexNAc5 1704 +++ Hex8HexNAc2 1743 −−− −−− +++ + +++ ++ ++Hex5HexNAc3dHex2 1752 −−− +++ +++ +++ Hex6HexNAc3dHex 1768 +++ +++Hex7HexNAc3 1784 −−− −−− Hex4HexNAc4dHex2 1793 −− +++ −− +++Hex5HexNAc4dHex 1809 −−− −−− +++ − Hex6HexNAc4 1825 +++ Hex3HexNAc6dHex1891 +++ Hex9HexNAc2 1905 −−− −−− − + ++ ++ Hex5HexNAc4dHex2 1955 −−−−−− −− −− Hex10HexNAc2 2067 −−− − +++ Hex5HexNAc4dHex3 2101 − − − +++Hex5HexNAc5dHex2 2158 +++ +++ Hex6HexNAc5dHex 2174 +++ Hex6HexNAc5dHex32466 +++ Code for profiling results, when compared to the profile beforethe reaction; +++: new signal appears; ++: signal is significantlyincreased; +: signal is increased; −: signal is decreased; −−: signal issignificantly decreased; −−−: signal disappears; blank: no change.

TABLE 9 Exoglycosidase profiling of cord blood CD133+ and CD133− cellneutral N-glycan fraction. α-Man, β1,4-Gal, β1,3-Gal, and β-GlcNAc referto specific exoglycosidase enzymes as described in the text. α-Manβ1,4-Gal β1,3-Gal β-GlcNAc Proposed composition m/z CD 133+ CD 133− CD133+ CD 133− CD 133+ CD 133− CD 133+ CD 133− Hex2HexNAc 568 + + +++HexHexNAc2 609 +++ ++ −−− Hex3HexNAc 730 −−− −−− +++ ++ +++ ++ ++HexHexNAc2dHex 755 +++ ++ −−− −−− Hex2HexNAc2 771 + −− ++ ++ + + +Hex4HexNAc 892 −−− −−− + ++ ++ + Hex2HexNAc2dHex 917 −−− −− ++ ++ ++ +Hex3HexNAc2 933 −− + + − + Hex2HexNAc3 974 +++ Hex5HexNAc 1054 −−− −− +++ + ++ + Hex3HexNAc2dHex 1079 −−− −− ++ + + ++ Hex2HexNAc3dHex 1120 +++++ ++ + ++ + −−− Hex3HexNAc3 1136 +++ + + −−− Hex6HexNAc 1216 −−−− + + + Hex4HexNAc2dHex 1241 −−− −−− + Hex5HexNAc2 1257 −− −− −Hex3HexNAc3dHex 1282 −− Hex4HexNAc3 1298 ++ + + + Hex3HexNAc4 1339 +++−−− Hex7HexNAc 1378 −−− −−− − +++ + Hex5HexNAc2dHex 1403 −−− −−− −−− −Hex6HexNAc2 1419 −− −− −− − − −− Hex3HexNAc3dHex2 1428 +++ − −Hex4HexNAc3dHex 1444 − − − Hex5HexNAc3 1460 −−− − + + Hex3HexNAc4dHex1485 −− + + − −−− Hex4HexNAc4 1501 −−− +++ −−− Hex8HexNAc 1540 −−− −−−−−− ++ Hex3HexNAc5 1542 −−− + − −−− Hex6HexNAc2dHex 1565 −−− −−− +++Hex7HexNAc2 1581 −−− −− −− −− − −− Hex4HexNAc3dHex2 1590 −−− − − − − +Hex5HexNAc3dHex 1606 −−− −−− + −−− Hex6HexNAc3 1622 −−− −−− −−− −− −Hex4HexNAc4dHex 1647 −−− −−− − −−− Hex5HexNAc4 1663 −−− − −− − −Hex3HexNAc5dHex 1688 −−− + −−− −−− Hex9HexNAc 1702 + Hex4HexNAc5 1704−−− −−− Hex8HexNAc2 1743 −−− −−− −− −− − −− Hex5HexNAc3dHex2 1752 − +++Hex6HexNAc3dHex 1768 Hex4HexNAc4dHex2 1793 Hex5HexNAc4dHex 1809 −−− −−−−−− − − Hex6HexNAc4 1825 − −−− Hex5HexNAc5 1866 −−− −−− −−− −−−Hex3HexNAc6dHex 1891 −−− Hex9HexNAc2 1905 −−− −−− −− −− − −−Hex6HexNAc3dHex2 1914 −−− −−− Hex5HexNAc4dHex2 1955 −− − −−−Hex6HexNAc4dHex 1971 −−− −−− −−− Hex7HexNAc4 1987 −−− −−−Hex5HexNAc5dHex 2012 +++ Hex6HexNAc5 2028 −−− −−− −−− Hex10HexNAc2 2067−−− −−− − − Hex5HexNAc4dHex3 2101 − − − Hex6HexNAc4dHex2 2117 −−− −−−−−− −−− Hex7HexNAc4dHex 2133 −−− Hex6HexNAc5dHex 2174 −−− −−− −−−Hex5HexNAc6dHex 2215 −−− Hex6HexNAc4dHex3 2263 −−− −−− Hex6HexNAc5dHex22320 −−− Hex6HexNAc5dHex3 2466 −−− Code for profiling results, whencompared to the profile before the reaction; +++: new signal appears;++: signal is significantly increased; +: signal is increased; −: signalis decreased; −−: signal is significantly decreased; −−−: signaldisappears; blank: no change.

TABLE 10 Exoglycosidase profiling of cord blood Lin+ and Lin− cellneutral N-glycan fraction. α-Man β1,4-Gal β1,3-Gal β-GlcNAc Proposedcomposition m/z LIN+ LIN− LIN+ LIN− LIN+ LIN− LIN+ LIN− Hex2HexNAc 568−−− +++ + + − HexHexNAc2 609 +++ +++ +++ Hex2HexNAcdHex 714 +++Hex3HexNAc 730 −−− +++ ++ +++ + +++ + HexHexNAc2dHex 755 +++ +++ + + +++Hex2HexNAc2 771 + + + + + + Hex4HexNAc 892 −−− −−− ++ + ++ + +Hex2HexNAc2dHex 917 −− −−− + ++ − − Hex3HexNAc2 933 − + + + − +Hex2HexNAc3 974 +++ Hex5HexNAc 1054 −− −−− ++ − − Hex3HexNAc2dHex 1079−− −−− ++ − ++ ++ Hex4HexNAc2 1095 −− −−− − Hex2HexNAc3dHex 1120 +++Hex3HexNAc3 1136 +++ + + + − +++ −−− Hex6HexNAc 1216 − −−− + + + +Hex4HexNAc2dHex 1241 −−− −−− + + −−− Hex5HexNAc2 1257 −− −−− ++ − − − +Hex3HexNAc3dHex 1282 + −− −−− Hex4HexNAc3 1298 + Hex2HexNAc4dHex 1323+++ +++ Hex3HexNAc4 1339 −−− ++ + −− −−− Hex7HexNAc 1378 −−− −−− + ++Hex5HexNAc2dHex 1403 −−− −−− + Hex6HexNAc2 1419 −− −− −− − − −Hex3HexNAc3dHex2 1428 +++ −−− −−− +++ Hex4HexNAc3dHex 1444 −−− − + +Hex5HexNAc3 1460 −−− Hex3HexNAc4dHex 1485 −− −−− −−− Hex4HexNAc4 1501 +−−− + − −−− −− −−− −−− Hex8HexNAc 1540 −−− −−− −−− + ++ Hex3HexNAc5 1542+++ ++ + ++ − Hex6HexNAc2dHex 1565 −−− −−− −−− Hex7HexNAc2 1581 −− −−−−− −− − Hex4HexNAc3dHex2 1590 − +++ Hex5HexNAc3dHex 1606 −−− −−− − −−−−−− −−− Hex2HexNAc4dHex3 1615 +++ Hex6HexNAc3 1622 −−− −−− −−− −−−Hex4HexNAc4dHex 1647 −−− −− −−− −−− −−− Hex5HexNAc4 1663 −−− −− −− − −−− Hex3HexNAc5dHex 1688 − −−− −−− Hex9HexNAc 1702 −−− −−− Hex4HexNAc51704 +++ −−− Hex8HexNAc2 1743 −− −−− −− −− − Hex5HexNAc3dHex2 1752 −−−+++ Hex6HexNAc3dHex 1768 −−− Hex3HexNAc4dHex3 1777 +++ Hex7HexNAc3 1784−−− Hex4HexNAc4dHex2 1793 +++ Hex5HexNAc4dHex 1809 + −−− −− −−− −−Hex6HexNAc4 1825 +++ − −−− −− +++ Hex4HexNAc5dHex 1850 +++ +++Hex5HexNAc5 1866 +++ −−− Hex3HexNAc6dHex 1891 −−− − Hex9HexNAc2 1905 −−−−−− −− −− − Hex4HexNAc4dHex3 1939 +++ Hex5HexNAc4dHex2 1955 −−− +++Hex6HexNAc4dHex 1971 −−− Hex7HexNAc4 1987 −−− +++ Hex5HexNAc5dHex 2012+++ −−− Hex6HexNAc5 2028 −−− Hex10HexNAc2 2067 −−− −−− − ++ +Hex5HexNAc4dHex3 2101 +++ Hex8HexNAc4 2149 −−− Hex6HexNAc5dHex 2174 −−−− Hex5HexNAc6dHex 2215 −−− −−− Hex11HexNAc2 2229 +++ Hex6HexNAc6 2231−−− −−− Hex6HexNAc5dHex2 2320 −−− −−− Hex12HexNAc2 2391 +++ +++ +++Hex7HexNAc6 2393 −−− −−− Hex6HexNAc5dHex3 2466 −−− −−− Hex7HexNAc6dHex2539 +++

TABLE 11 Differential effect of α2,3-sialidase treatment on isolatedsialylated N-glycans from cord blood CD133⁺ and CD133⁻ cells. Theneutral N-glycan columns show that neutral N-glycans corresponding tothe listed sialylated N-glycans appear in analysis of CD133⁺ cellN-glycans but not CD133⁻ cell N-glycans. Proposed glycan compositionsoutside parenthesis are visible in the neutral N-glycan fraction afterα2,3-sialidase digestion of CD133⁺ cell sialylated N-glycans. SialylatedNeutral N-glycan N-glycan m/z Proposed monosaccharide composition CD133⁺CD133⁻ CD133⁺ CD133⁻ 1768 (NeuAc₁)Hex₄HexNAc₄ + + + − 2156(NeuAc₁)Hex₈HexNAc₂dHex₁/ + + + − (NeuAc₁Hex₅HexNAc₄dHex₁SO₃) 2222(NeuAc₁)Hex₅HexNAc₄dHex₂ + + + − 2238 (NeuAc₁Hex₆HexNAc₄dHex₁/ + + + −(NeuGc₁)Hex₅HexNAc₄dHex₂ 2254 (NeuAc₁)Hex₇HexNAc₄/ + + + −(NeuGc₁)Hex₆HexNAc₄dHex₁ 2368 (NeuAc₁)Hex₅HexNAc₄dHex₃ + + + − 2447(NeuAc₂)Hex₈HexNAc₂dHex₁/ + + + − (NeuAc₂Hex₅HexNAc₄dHex₁SO₃) 2448(NeuAc₁)Hex₈HexNAc₂dHex₃/ + + + − (NeuAc₁Hex₅HexNAc₄dHex₃SO₃) 2513(NeuAc₂)Hex₅HexNAc₄dHex₂ + + + − 2733 (NeuAc₁)Hex₆HexNAc₅dHex₃ + + + −2953 (NeuAc₁)Hex7HexNAc₆dHex₂ + + + −

TABLE 12 Proposed neutral N-glycan grouping of the samples; hESC, humanembryonal stem cell line, lines 1-4, EB, embryoid bodies derived fromhESC lines 3 and 4, st.3 3, stage 3 differentiated cells from hESC line3, HEF human fibroblasts used as feeder cells. Neutral N-glycanGrouping: Composition Glycan Grouping hESC 1 hESC 2 hESC 3 hESC 4 EB 3EB 4 st.3 3 HEF1 HEF2 General N-glycan grouping: Hex₅₋₁₂HexNAc₂high-mannose 84.4 73.2 80.0 79.0 64.4 79.1 73.6 82.6 77.5Hex₁₋₄HexNAc₂dHex₀₋₁ low-mannose 5.6 10.9 6.8 7.8 11.5 9.2 9.4 7.1 8.0n_(HexNAc) = 3 and n_(Hex) ≧ 2 hybrid/monoantennary 3.4 6.7 3.2 3.2 9.06.7 6.5 5.4 5.1 n_(HexNAc) ≧ 4 and n_(Hex) ≧ 2 complex 6.2 8.9 10.1 10.014.5 5.0 10.3 4.9 9.1 Other types 0.3 0.3 0.0 0.0 0.7 0.0 0.3 0.0 0.2Complex/hybrid/monoantennary N-glycan grouping: n_(dHex) ≧ 1 fucosylated52.3 40.4 65.3 62.4 46.1 27.9 36.9 51.6 56.6 n_(dHex) ≧ 2 α2/3/4-linkedFuc 11.7 1.8 11.7 13.9 6.9 9.9 2.2 0.0 3.4 n_(HexNAc) > n_(Hex) ≧ 2terminal HexNAc 9.4 17.4 6.8 6.0 17.7 15.5 18.4 27.2 16.2 n_(HexNAc) =n_(Hex) ≧ 5 bisecting GlcNAc 0.0 10.2 0.0 0.0 7.8 4.2 9.7 0.0 0.0Complex N-glycan grouping: n_(HexNAc) ≧ 5 and n_(Hex) ≧ 6 largeN-glycans 11.3 5.4 13.7 8.7 3.3 0.0 4.6 14.1 20.5

TABLE 13 Proposed sialylated N-glycan grouping of the samples; hESC,human embryonal stem cell line, lines 2-4, EB, embryoid bodies derivedfrom hESC line 3, st.3 3, stage 3 differentiated cells from hESC line 3,HEF human fibroblasts used as feeder cells. Sialylated N-glycanGrouping: Composition Glycan Grouping hESC 2 hESC 3 hESC 4 EB 3 st.3 3hEF General N-glycan grouping: n_(HexNAc) = 3 and nHex ≧ 5 hybrid 0.03.8 4.5 9.6 3.6 3.4 n_(HexNAc) = 3 and n_(Hex) = 3 or 4 monoantennary2.2 2.3 5.5 6.4 2.5 3.6 n_(HexNAc) ≧ 4 and n_(Hex) ≧ 3 complex 97.8 92.689.1 79.1 93.9 92.2 Other types — 0.0 1.3 0.9 4.8 0.0 0.8Complex/hybrid/monoantennary N-glycan grouping: n_(dHex) ≧ 1 fucosylated93.0 72.6 74.6 79.3 85.3 76.2 n_(dHex) ≧ 2 α2/3/4-linked Fuc 33.5 23.018.5 10.8 5.2 20.4 n_(HexNAc) > n_(Hex) ≧ 3 terminal HexNAc 7.8 6.4 5.27.7 3.0 0.8 n_(HexNAc) = n_(Hex) ≧ 5 bisecting GlcNAc 4.3 3.9 2.2 12.525.8 1.4 n_(NeuGc) ≧ 1 NeuGc-containing 0.0 6.8 5.6 1.5 0.0 0.0 ComplexN-glycan grouping: n_(HexNAc) ≧ 5 and n_(Hex) ≧ 6 large N-glycans 22.718.7 14.9 12.4 26.6 44.5 sialylation degree SD_(HexNAc) = 51.6 60.4 63.060.7 56.6 60.3 n_(NeuAc/Gc):(n_(HexNAc) − 2)

TABLE 14 Mass spectrometric analysis results of sialylated N-glycanswith monosaccharide compositions NeuAc₁₋₂Hex₅HexNAc₄dHex₀₋₃ insequential enzymatic modification steps of human cord blood mononuclearcells. The columns show relative glycan signal intensities (% of thetabled signals) before the modification reactions (MNC), afterα2,3-sialyltransferase reaction (α2,3SAT), and after sequentialα2,3-sialyltransferase and α1,3-fucosyltransferase reactions (α2,3SAT +α1,3FucT). The sum of the glycan signal intensities in each column hasbeen normalized to 100% for clarity. calc m/z Proposed monosaccharide [M− α2,3SAT + composition H]⁻ MNC α2,3SAT α1,3FucT NeuAcHex5HexNAc41930.68 24.64 12.80 13.04 NeuAcHex5HexNAc4dHex 2076.74 39.37 30.11 29.40NeuAcHex5HexNAc4dHex2 2222.8 4.51 8.60 6.83 NeuAcHex5HexNAc4dHex32368.85 3.77 6.34 6.45 NeuAc2Hex5HexNAc4 2221.78 13.20 12.86 17.63NeuAc2Hex5HexNAc4dHex 2367.83 14.04 29.28 20.71 NeuAc2Hex5HexNAc4dHex22513.89 0.47 n.d. 5.94

TABLE 15 Mass spectrometric analysis results of selected neutralN-glycans in enzymatic modification steps of human cord bloodmononuclear cells. The columns show relative glycan signal intensities(% of the total glycan signals) before the modification reactions (MNC),after broad-range sialidase reaction (SA'se), afterα2,3-sialyltransferase reaction (α2,3SAT), after α1,3-fucosyltransferase reaction (α1,3FucT), and after sequentialα2,3-sialyltransferase and α1,3- fucosyltransferase reactions (α2,3SAT +α1,3FucT). calc m/z α2,3SAT + Proposed monosaccharide composition [M +H]⁺ MNC SA'ase α2,3SAT α1,3FucT α1,3FucT Hex5HexNAc2 1257.42 11.94 14.1114.16 13.54 9.75 Hex3HexNAc4dHex 1485.53 0.76 0.63 0.78 0.90 0.78Hex6HexNAc3 1622.56 0.61 1.99 0.62 0.51 0.40 Hex5HexNAc4 1663.58 0.444.81 0.00 0.06 0.03 Hex5HexNac4dHex 1809.64 0.19 1.43 0.00 0.25 0.00Hex5HexNac4dHex2 1955.7 0.13 0.22 0.00 0.22 0.00 Hex6HexNAc5 2028.710.07 1.14 0.00 0.00 0.00 Hex5HexNAc4dHex3 2101.76 0.12 0.09 0.00 0.220.00 Hex6HexNAc5dHex 2174.77 0.00 0.51 0.00 0.14 0.00 Hex6HexNAc5dHex22320.83 0.00 0.00 0.00 0.08 0.00

TABLE 16 Cord blood mononuclear cell sialylated N-glycan signals. Them/z values refer to monoisotopic masses of [M − H]⁻ ions. Proposedmonosaccharide composition m/z (calculated) NeuAcHex3HexNAc3dHex 1549.551549 NeuAcHex4HexNAc3 1565.55 1565 NeuAc2Hex3HexNAc2dHex 1637.57 1637NeuAc2Hex2HexNAc3dHex 1678.60 1678 NeuAcHex4HexNAc3dHex 1711.61 1711NeuAcHex5HexNAc3 1727.60 1727 NeuAcHex3HexNAc4dHex 1752.63 1752NeuAcHex4HexNAc4 1768.57 1768 NeuAcHex4HexNAc3dHexSO3 1791.56 1791NeuAc2Hex3HexNAc3dHex 1840.65 1840 NeuAcHex4HexNAc3dHex2 1857.66 1857Hex5HexNAc4dHexSO3 1865.60 1865 NeuAcHex5HexNAc3dHex 1873.66 1873NeuAcHex6HexNAc3 1889.65 1889 NeuAcHex3HexNAc4dHex2 1898.69 1898NeuAcHex4HexNAc4dHex 1914.68 1914 NeuAcHex5HexNAc4 1930.68 1930NeuAc2Hex4HexNAc3dHex/ 2002.70 2002 Hex8HexNAc3SO3 NeuAc2Hex5HexNAc32018.70 2018 NeuAcHex6HexNAc3dHex 2035.71 2035 NeuAcHex7HexNAc3 2051.712051 Hex4HexNAc5dHex2SO3 2052.68 2052 NeuAc2Hex4HexNAc4 2059.72 2059NeuAcHex4HexNAc4dHex2 2060.74 2060 NeuAcHex5HexNAc4dHex 2076.74 2076NeuAcHex6HexNAc4 2092.73 2092 NeuAcHex4HexNAc5dHex 2117.76 2117NeuAcHex5HexNAc5 2133.76 2133 NeuAcHex8HexNAc2dHex/ 2156.74/2156.69 2156NeuAcHex5HexNAc4dHexSO3 NeuAc2Hex5HexNAc4 2221.78 2221NeuAcHex5HexNAc4dHex2 2222.80 2222 Hex6HexNAc5dHexSO3 2230.73 2230NeuAcHex6HexNAc4dHex/ 2238.79 2238 NeuGcHex5HexNAc4dHex2NeuAcHex7HexNAc4/ 2254.79 2254 NeuGcHex6HexNAc4dHex NeuAcHex5HexNAc5dHex2279.82 2279 NeuAc2Hex4HexNAc3dHex3 2294.82 2294 NeuAcHex6HexNAc52295.81 2295 NeuAc2Hex5HexNAc4dHex 2367.83 2367 NeuAcHex5HexNAc4dHex32368.85 2368 NeuAc2Hex6HexNAc4 2383.83 2383 NeuAcHex6HexNAc4dHex22384.85 2384 NeuAc2Hex5HexNAc3dHexSO3 2390.77 2390NeuAc2Hex3HexNAc5dHex2 2392.86 2392 NeuAcHex5HexNAc5dHex2 2425.87 2425NeuAcHex6HexNAc5dHex 2441.87 2441 NeuAc2Hex8HexNAc2dHex/ 2447.83/2447.792447 NeuAc2Hex5HexNAc4dHexSO3 NeuAcHex7HexNAc5 2457.86 2457NeuAc2Hex5HexNAc4dHex2 2513.89 2513 NeuAcHex6HexNAc5dHexSO3 2521.83 2521NeuAcHex6HexNAc4dHex3 2530.91 2530 NeuAc3Hex4HexNAc5 2553.90 2553NeuAc2Hex5HexNAc5dHex 2570.91 2570 NeuAcHex5HexNAc5dHex3 2571.93 2571NeuAc2Hex6HexNAc5 2586.91 2586 NeuAcHex6HexNAc5dHex2 2587.93 2587Hex7HexNAc6dHexSO3 2595.86 2595 NeuAcHex7HexNAc5dHex 2603.92 2603NeuAcHex6HexNAc6dHex 2644.95 2644 NeuAcHex7HexNAc6 2660.94 2660NeuAc2Hex4HexNAc5dHex2(SO3)2 2714.83 2714 NeuAc2Hex6HexNAc5dHex 2732.972732 NeuAcHex6HexNAc5dHex3 2733.99 2733 NeuAcHex7HexNAc6dHex 2807.002807 NeuAcHex6HexNAc5dHex3SO3 2813.94 2813 NeuAc3Hex6HexNAc5 2878.002878 NeuAc2Hex6HexNAc5dHex2 2879.02 2879 NeuAcHex6HexNAc5dHex4 2880.042880 NeuAc2Hex5HexNAc6dHex2 2920.05 2920 NeuAc2Hex7HexNAc6 2952.04 2952NeuAcHex7HexNAc6dHex2 2953.06 2953 NeuAcHex7HexNac7dHex 3010.08 3010NeuAc3Hex6HexNAc5dHex 3024.06 3024 NeuAc2Hex6HexNAc5dHex3 3025.09 3025NeuAcHex8HexNAc7 3026.08 3026 NeuAc2Hex7HexNAc6dHex 3098.10 3098NeuAcHex7HexNAc6dHex3 3099.12 3099 NeuAc2Hex6HexNAc5dHex4 3171.14 3171NeuAcHex8HexNAc7dHex 3172.13 3172

TABLE 17 NMR analysis of hESC neutral N-glycans (hESC sample). Referenceglycans (A.-D.) are described in FIG. 26. B C D hESC sample Glycanresidue linkage A proton ppm ppm ppm ppm ppm D-GlcNAc H-1a 5.191 5.1875.187 5.188 5.188 H-1b 4.690 4.693 4.693 4.695 4.694 NAc 2.042 2.0372.037 2.038 2.038 β-D-GlcNAc 4 H-1 4.596 4.586 4.586 4.600 4.596 NAc2.072 2.063 2.063 2.064 2.061¹⁾ β-D-Man 4, 4 H-1 4.775 4.771 4.771 4.780H-2 4.238 4.234 4.234 4.240 4.234 α-D-Man 6, 4, 4 H-1 4.869 4.870 4.8704.870 4.869 H-2 4.149 4.149 4.149 4.150 4.153 α-D-Man 6, 6, 4, 4 H-15.153 5.151 5.151 5.143 5.148 H-2 4.025 4.021 4.021 4.020 4.023 α-D-Man2, 6, 6, 4, 4 H-1 5.047 5.042 5.042 5.041 5.042 H-2 4.074 4.069 4.0694.070 4.069 α-D-Man 3, 6, 4, 4 H-1 5.414 5.085 5.415 5.092 5.408, 5.085H-2 4.108 4.069 4.099 4.070 4.102, 4.069 α-D-Man 2, 3, 6, 4, 4 H-1 5.047— 5.042 — 5.042 H-2 4.074 — 4.069 — 4.069 α-D-Man 3, 4, 4 H-1 5.3435.341 5.341 5.345 5.346, 5.338 H-2 4.108 4.099 4.099 4.120 4.102 α-D-Man2, 3, 4, 4 H-1 5.317 5.309 5.050 5.055 5.310, 5.057 H-2 4.108 4.0994.069 4.070 4.102, 4.069 α-D-Man 2, 2, 3, 4, 4 H-1 5.047 5.042 — — 5.042H-2 4.074 4.069 — — 4.069 ¹⁾Under HDO.

TABLE 18 NMR analysis of hESC acidic N-glycans (hESC sample). Referenceglycans (A.-E.) are described in FIG. 27. Glycan A. B. C. D. E. hESCsample residue linkage proton ppm ppm ppm ppm ppm ppm D-GlcNAc H-1a5.180 5.188 5.189 5.181 5.189 5.182/5.188 H-1b 4.692 n.a.¹⁾ 4.695 n.a.4.694 n.a. NAc 2.038 2.038 2.038 2.039 2.038 2.038 α-L-Fuc 6 H-1a 4.890—²⁾ — 4.892 — 4.893 H-1b 4.897 — — 4.900 — 4.893 H-5a 4.098 — — 4.10  —Overlap³⁾ H-5b 4.134 — — n.a. — Overlap CH3a 1.209 — — 1.211 — 1.210CH3b 1.220 — — 1.223 — 1.219 β-D-GlcNAc 4 H-1a 4.664 4.612 4.614 4.6634.613 n.a. H-1b 4.669 4.604 4.606 n.a. 4.604 n.a./4.605 NAc 2.097 2.0812.081 2.096/ 2.084 2.081/2.095 (a/b) 2.093 β-D-Man 4, 4 H-1 4.772 n.a.n.a. n.a. n.a. n.a H-2 4.257 4.246 4.253 4.248 4.258 4.256 α-D-Man 6, 4,4 H-1 4.929 4.928 4.930 4.922 4.948 4.927 H-2 4.111 4.11  4.112 4.11 4.117 Overlap β-D-GlcpNAc 2, 6, 4, 4 H-1 4.583 4.581 4.582 4.573 4.6044.579/4.605 NAc 2.048 2.047 2.047 2.043 2.066 2.047/2.069 β-D-Gal 4, 2,6, 4, 4 H-1 4.544 4.473 4.473 4.550 4.447 4.447/4.472/ 4.545 H-3 n.a.n.a. n.a. 4.119 n.a. Overlap H-4 4.185 n.a. n.a. n.a. n.a. 4.185α-D-Galp 3, 4, 2, 6, 4, 4 H-1 5.146 — — — — 5.146 α-D-Neup5Ac 3, 4, 2,6, 4, 4 H-3a — — — 1.800 — 1.802 H-3e — — — 2.758 — 2.756 NAc — — —2.031 — 2.030 α-D-Neup5Ac 6, 4, 2, 6, 4, 4 H-3a — — — — 1.719 1.721 H-3e— — — — 2.673 2.669 NAc — — — — 2.029 2.030 α-D-Man 3, 4, 4 H-1 5.1355.118 5.135 5.116 5.133 5.118/5.134 H-2 4.195 4.190 4.196 4.189 4.1974.195 β-D-GlcpNAc 2, 3, 4, 4 H-1 4.605 4.573 4.606 4.573 4.6044.579/4.605 NAc 2.069 2.047 2.069 2.048 2.070 2.047/2.069 β-D-Galp 4, 2,3, 4, 4 H-1 4.445 4.545 4.445 4.544 4.443 4.445/4.545 H-3 n.a. 4.113n.a. 4.113 n.a. Overlap α-D-Neup5Ac 6, 4, 2, 3, 4, 4 H-3a 1.722 — 1.719— 1.719 1.721 H-3e 2.666 — 2.668 — 2.667 2.669 NAc 2.029 — 2.030 — 2.0292.030 α-D-Neup5Ac 3, 4, 2, 3, 4, 4 H-3a — 1.797 — 1.797 — 1.802 H-3e —2.756 — 2.758 — 2.756 NAc — 2.030 — 2.031 — 2.030 ¹⁾n.a., not assigned.²⁾—, not present. ³⁾Overlap, overlapping signals at 4.139-4.088 ppm.

TABLE 19 Detected neutral O-glycan fraction signals from CB MNC. NeutralO-glycan signals, [M + Na]⁺ ions Proposed structure calc. m/z exp. m/zHex1HexNAc2 611.23 611.19 Hex2HexNAc2 773.28 773.29 Hex4HexNAc2 1097.391097.44 Hex3HexNAc3 1138.42 1138.47 Hex5HexNAc2 1259.44 1259.5Hex3HexNAc4 1341.50 1341.56 Hex5HexNAc3 1462.52 1462.62 Hex4HexNAc41503.55 1503.63 Hex3HexNAc3dHex4 1722.65 1722.71 Hex4HexNAc3dHex41884.70 1884.77 Hex5HexNAc5dHex1 2014.74 2014.86 Hex4HexNAc6dHex12055.77 2055.85 Hex6HexNAc5dHex1 2176.79 2176.89

TABLE 20 Detected acidic O-glycan fraction signals from CB MNC. AcidicO-glycan signals, [M − H]⁻ ions Proposed structure calc. m/z exp. m/zNeuAc1Hex1HexNAc1 675.25 675.27 NeuAc2Hex1HexNAc1 966.35 966.37NeuAc1Hex2HexNAc2 1040.38 1040.54 NeuAc1Hex2HexNAc2dHex1 1186.44 1186.47NeuGc1Hex3HexNAc2 1218.43 1218.48 NeuAc2Hex2HexNAc2 1331.48 1331.61NeuAc1Hex3HexNAc3 1405.51 1405.75 NeuAc2Hex2HexNAc1dHex1 1477.54 1477.65NeuAc2Hex3HexNAc3 1696.61 1696.78 NeuAc1Hex3HexNAc3dHexSP2 1711.491711.91 NeuAc1Hex4HexNAc4 1770.59 1770.97 NeuAc1Hex5HexNAc4 1932.701932.89 NeuAc1Hex4HexNAc4dHex1(SP)2 2076.61 2076.98 NeuAc2Hex5HexNAc42223.80 2224.00

TABLE 21 Detected glycan signals in the neutral O-glycan fraction fromhESC. Neutral O-glycan reducing oligosaccharides, [M + Na]⁺ ionsProposed structure calc. m/z exp. m/z Hex1HexNAc2 609.21 609.26Hex3HexNAc1 730.24 730.30 Hex2HexNAc2 771.26 771.33NeuAc1Hex1HexNAc1(deoxyamino)HexNAc1 899 899.39 Hex2HexNAc2dHex1 917.32917.40 Hex3HexNAc2 933.31 933.39 Hex2HexNAc3 974.34 974.44Hex2HexNAc2dHex2 1063.38 1063.46 Hex3HexNAc2dHex1 1079.38 1079.44Hex4HexNAc2 1095.37 1095.45 Hex3HexNAc3 1136.40 1136.47 Hex5HexNAc21257.42 1257.49 Hex3HexNAc3dHex1 1282.45 1282.52 Hex4HexNAc3 1298.451298.52 Hex7HexNAc1 1378.45 1378.52 Hex6HexNAc2 1419.48 1419.54Hex4HexNAc3dHex1 1444.51 1444.57 Hex5HexNAc3 1460.50 1460.56Hex3HexNAc4dHex1 1485.53 1485.6 Hex3HexNAc5 1542.56 1542.58 Hex7HexNAc21581.53 1581.59 Hex6HexNAc3 1622.56 1622.61 Hex4HexNAc4dHex1 1647.591647.63 Hex4HexNAc5 1704.61 1704.66 Hex8HexNAc2 1743.58 1743.63Hex5HexNAc4dHex1 1809.64 1809.69 Hex5HexNAc5 1866.66 1866.70 Hex9HexNAc21905.63 1905.68 Hex10HexNAc2 2067.69 2067.72

TABLE 22 Detected acidic O-glycan signals from hESC. Acidic O-glycanreducing oligosaccharides, [M − H]⁻ ions Proposed structure calc. m/zexp. m/z NeuAc2HexHexNAc 964.33 964.35 SaHex2HexNAc2 1038.36 1038.49NeuAcHex2HexNAc2dHex 1184.42 1184.5 Hex3HexNAc3SP 1192.36 1192.73SaHex3HexNAc2 1200.42 1200.43 NeuAc2Hex2HexNAc2/ 1329.46 1329.56NeuGcNeuAcHexHexNAc2dHex Hex3HexNAc3dHexSP 1338.41 1338.6 SaHex3HexNAc31403.49 1403.62 Sa2Hex2HexNAcdHex 1475.52 1475.79NeuAcHex6HexNAc/NeuAcHex3HexNAc3SP 1483.49 1483.71 SaHex3HexNAc3dHex1549.55 1549.9 Hex4HexNAc4SP 1557.49 1557.72 SaHex4HexNAc3 1565.551565.66 NeuAc2Hex3HexNAc3 1694.59 1694.8 Hex4HexNAc4dHexSP 1703.551703.9 SaHex4HexNAc3dHex 1711.61 1711.78 SaHex5HexNAc3 1727.60 1727.96SaHex4HexNAc4 1768.57 1768.75 SaHex6HexNAc3 1889.65 1889.96SaHex4HexNAc4dHex 1914.68 1915.04 SaHex5HexNAc4 1930.68 1930.83SaHex5HexNAc4dHex 2076.74 2076.91 NeuGcHex5HexNAc4dHex/SaHex6HexNAc42092.73 2092.86 Sa2Hex5HexNAc4 2221.78 2221.82 SaHex5HexNAc4dHex22222.80 2222.93 NeuGcHex5HexNAc4dHex2/ 2238.79 2238.9 SaHex6HexNAc4dHexSaHex7HexNAc4/NeuGcHex6HexNAc4dHex 2254.79 2254.88 SaHex5HexNAc4dHex32368.85 2368.26 SaHex6HexNAc5dHex 2441.87 2442.23

TABLE 23 Exoglycosidase analysis results of hESC line FES 29 grown onmEF. FES 29 Proposed composition m/z α-Man β-GlcNAc β-HexNAc β1,4Galβ1,3-Gal α1,3/4-Fuc α1,2-Fuc Hex2HexNAc 568 +++ +++ +++ +++ +++HexHexNAc2 609 +++ +++ +++ Hex3HexNAc 730 −− + ++ ++ + + HexHexNAc2dHex755 +++ Hex2HexNAc2 771 + + −−− + + + Hex4HexNAc 892 −−− + + −−− + + +Hex2HexNAc2dHex 917 −− + + + + Hex3HexNAc2 933 −− ++ + + + + Hex2HexNAc3974 +++ +++ +++ +++ Hex5HexNAc 1054 −− + + + + + + Hex3HexNAc2dHex 1079−− ++ + + + + Hex4HexNAc2 1095 −− + + + + Hex2HexNAc3dHex 1120 ++ − + −−Hex3HexNAc3 1136 + −− −− + ++ + Hex6HexNAc 1216 −− + ++ + + + +Hex4HexNAc2dHex 1241 −− + + Hex5HexNAc2 1257 −− Hex3HexNAc3dHex 1282 −−− + + + Hex4HexNAc3 1298 + ++ ++ ++ + ++ ++ Hex3HexNAc4 1339 −−− −−− ++++ −−− −−− Hex7HexNAc 1378 −− + + + + + + Hex5HexNAc2dHex 1403 −−− + +Hex6HexNAc2 1419 −− − − − − Hex3HexNAc3dHex2 1428 +++ +++Hex4HexNAc3dHex 1444 ++ + + + Hex5HexNAc3 1460 − + + + Hex3HexNAc4dHex1485 −− −−− + + + Hex4HexNAc4 1501 −−− −−− −−− + −−− −−− Hex8HexNAc 1540−−− + ++ + + Hex3HexNAc5 1542 ++ −−− −−− ++ ++ + Hex6HexNAc2dHex 1565−−− −−− Hex7HexNAc2 1581 −− − Hex4HexNAc3dHex2 1590 ++ + Hex5HexNAc3dHex1606 − + + Hex6HexNAc3 1622 −− −− + + + Hex4HexNAc4dHex 1647 −− −−−−− + + Hex5HexNAc4 1663 + − + + Hex3HexNAc5dHex 1688 −−− −−− + +Hex9HexNAc 1702 −−− + ++ + + Hex4HexNAc5 1704 + − −−− −−− Hex8HexNAc21743 −−− − − − − Hex5HexNAc3dHex2 1752 +++ Hex6HexNAc3dHex 1768 −− −−Hex7HexNAc3 1784 −− −− + Hex4HexNAc4dHex2 1793 −−− −−− −−− ++ −−−Hex5HexNAc4dHex 1809 − + + + Hex6HexNAc4 1825 −− Hex4HexNAc5dHex 1850−−− −−− −−− ++ Hex5HexNAc5 1866 + + ++ ++ ++ Hex3HexNAc6dHex 1891 ++++++ +++ Hex9HexNAc2 1905 −−− − − − − − Hex7HexNAc3dHex 1930 +++Hex5HexNAc4dHex2 1955 − Hex6HexNAc4dHex 1971 −− Hex7HexNAc4 1987 + +Hex4HexNAc5dHex2 1996 −−− −−− −−− Hex5HexNAc5dHex 2012 −−− −−− +Hex6HexNAc5 2028 − Hex10HexNAc2 2067 −−− + + + + Hex5HexNAc6 2069 +++Hex5HexNAc4dHex3 2101 −− Hex6HexNAc4dHex2 2117 +++ +++ Hex7HexNAc4dHex2311 Hex4HexNAc5dHex3 2142 +++ +++ +++ Hex8HexNAc4 2149 +++Hex5HexNAc5dHex2 2158 +++ +++ Hex6HexNAc5dHex 2174 −− Hex3HexNAc6dHex32183 +++ +++ +++ Hex7HexNAc5 2190 Hex11HexNAc2 2229 −−− Hex6HexNAc6 2231+++ Hex5HexNAc4dHex4 2247 +++ Hex7HexNAc4dHex2 2279 +++ +++ +++Hex5HexNAc5dHex3 2304 +++ +++ Hex6HexNAc5dHex2 2320 +++ +++ +++ +++ +++Hex7HexNAc5dHex 2336 − Hex8HexNAc5 2352 −−− Hex12HexNAc2 2391 −−−Hex7HexNAc6 2393 +++ +++ Hex7HexNAc4dHex3 2425 +++ +++ Hex6HexNAc5dHex32466 +++ +++ Hex8HexNAc5dHex 2498 −−− Hex9HexNAc5 2514 Hex7HexNAc6dHex2539 +++ +++ +++ Hex13HexNAc2 2553 +++ Hex8HexNAc6 2555 +++ +++Hex9HexNAc5dHex 2660 Hex7HexNAc6dHex4 2978 +++ Hex8HexNAc6dHex4 3140 ++++++ Hex9HexNAc6dHex4 3302 +++ +++ +++ Hex10HexNAc6dHex4 3464 +++ +++ +++Hex11HexNAc6dHex4 3626 +++ +++ +++ Hex12HexNAc6dHex4 3788 +++ +++

TABLE 24 Exoglycosidase analysis results of hESC line FES 29 (st 1)grown on hEF and cmbryoid bodies (EB, st 2). FES 29 st 1 FES 29 st 2 FES29 st 1 FES 29 st 2 Proposed composition m/z α-Man α-Man β1,4-Galβ1,4-Gal HexHexNAc2 609 ++ ++ −−− −− HexHexNAc2dHex 755 +++ +++Hex2HexNAc2 771 +++ ++ Hex4HexNAc 892 −−− Hex2HexNAc2dHex 917 −−− −−−Hex3HexNAc2 933 ++ ++ + + Hex5HexNAc 1054 Hex3HexNAc2dHex 1079 −−− −− −Hex4HexNAc2 1095 −−− −− + + Hex2HexNAc3dHex 1120 + Hex3HexNAc3 1136 + ++++ ++ Hex6HexNAc 1216 Hex4HexNAc2dHex 1241 −−− −−− −−− Hex5HexNAc2 1257−− −− Hex3HexNAc3dHex 1282 ++ ++ Hex4HexNAc3 1298 + ++ + + Hex3HexNAc41339 +++ +++ Hex7HexNAc 1378 −−− −−− −−− Hex5HexNAc2dHex 1403 −−−Hex6HexNAc2 1419 −− −− Hex3HexNAc3dHex2 1428 +++ +++ Hex4HexNAc3dHex1444 − + + Hex5HexNAc3 1460 + + Hex3HexNAc4dHex 1485 ++ ++ Hex8HexNAc1540 −−− Hex3HexNAc5 1542 + +++ ++ Hex6HexNAc2dHex 1565 −−− −−−Hex7HexNAc2 1581 −− −− Hex5HexNAc3dHex 1606 −−− −−− − Hex6HexNAc3 1622−−− −− −−− −−− Hex4HexNAc4dHex 1647 − Hex5HexNAc4 1663 −−− −−−Hex3HexNAc5dHex 1688 −−− ++ ++ Hex9HexNAc 1702 Hex4HexNAc5 1704 +++ −−Hex8HexNAc2 1743 −− −− Hex6HexNAc3dHex 1768 Hex4HexNAc4dHex2 1793 +++Hex5HexNAc4dHex 1809 − −− −− Hex4HexNAc5dHex 1850 −−− −− Hex5HexNAc51866 −−− Hex3HexNAc6dHex 1891 +++ Hex9HexNAc2 1905 −−− −−−Hex5HexNAc4dHex2 1955 − − −−− Hex6HexNAc4dHex 1971 −−− Hex4HexNAc5dHex21996 −−− −−− −−− Hex5HexNAc5dHex 2012 −−− Hex6HexNAc5 2028 −−−Hex10HexNAc2 2067 −−− −−− Hex5HexNAc4dHex3 2101 − Hex4HexNAc5dHex3 2142−−− −−− Hex5HexNAc5dHex2 2158 −−− −−− Hex6HexNAc5dHex 2174 −−− −−−Hex11HexNAc2 2229 ++ ++ Hex6HexNAc5dHex2 2320 −−− Hex12HexNAc2 2391 +++++ Hex13HexNAc2 2553 +++ +++ Hex14HexNAc2 2715 +++

TABLE 25 Exoglycosidase digestion analyses of hESC acidic N-glycans(cell line FES 29, grown on mEF). α3/4Fuc Proposed composition m/z α3SAα3/4Fuc →α2Fuc SA Hex3HexNAc2SP 989 + −−− −−− −−− NeuAcHex3HexNAc 997+++ Hex2HexNAc3SP 1030 + −−− −−− + Hex4HexNac2SP 1151 + −−− +Hex3HexNAc3SP 1192 ++ ++ ++ NeuAc2Hex2HexNAcdHex 1272 −−− −−− −−−Hex4HexNAc2dHexSP 1297 −−− −−− −−− + NeuAc2HexHexNAc2dHex 1313 + −−− ++Hex3HexNAc3dHexSP 1338 + −−− −−− ++ Hex4HexNAc3SP 1354 ++ + ++ ++Hex3HexNac4SP 1395 + + ++ NeuAcHex3HexNAc3 1403 + −−− NeuGcHex3HexNAc31419 −−− NeuAc2Hex2HexNAcdHex 1475 + + ++ Hex4HexNAc3dHexSP 1500 + +Hex5HexNAc3dHexSP/NeuAc2HexHexNAc3dHex 1516 + + Hex3HexNAc4dHexSP 1541 +++ ++ NeuAcHex3HexNAc3dHex 1549 + + + −−− Hex4HexNAc4SP 1557 ++ + ++NeuAcHex4HexNAc3 1565 − + −− NeuGcHex4HexNAc3 1581 + NeuAcHex3HexNAc41606 +++ NeuAc2Hex3HexNAc2dHex 1637 + + Hex4HexNAc3dHex2SP 1646 +++Hex5HexNAc3dHexSP 1662 + −−− −−− + NeuAc2Hex2HexNAc3dHex 1678 + − +NeuAcHex2HexNAc3dHex3 1679 +++ +++ Hex4HexNAc4dHexSP 1703 ++ ++ ++NeuAcHex4HexNAc3dHex 1711 + −− Hex5HexNAc4SP 1719 ++ + ++NeuAcHex5HexNAc3 1727 − − −− NeuGcHex5HexNAc3 1743 −−− + +NeuAcHex3HexNAc4dHex 1752 −−− −−− Hex4HexNAc5SP 1760 + + ++NeuAcHex4HexNAc4 1768 + + −− Hex7HexNAc2dHexSP 1783 NeuGcHex4HexNAc41784 +++ +++ +++ +++ Hex5HexNAc4SP2/NeuAc2Hex4HexNAc2dHex 1799 ++ ++Hex6HexNAc3dHexSP 1824 +++ +++ NeuAc2Hex3HexNAc3dHex 1840 + +NeuAcHex3HexNAc3dHex3 1841 +++ Hex5HexNAc4dHexSP 1865 ++ + ++NeuAcHex5HexNAc3dHex 1873 − − −−− Hex6HexNAc4SP 1881 ++ + −−− ++NeuAcHex6HexNAc3 1889 − −− Hex4HexNAc5dHexSP 1906 + + ++NeuAcHex4HexNAc4dHex 1914 − + + −− Hex5HexNAc5SP 1922 +++ +++NeuAcHex5HexNAc4 1930 + + + −− NeuGcHex5HexNAc4 1946 ++ + ++NeuAcHex3HexNAc5dHex 1955 + −−− −−−NeuAc2Hex5HexNAc2dHex/Hex6HexNAc4(SP)2 1961 +++ NeuAcHex4HexNAc51971 + + NeuAc2Hex4HexNAc3dHex/Hex8HexNAc3SP 2002 + −NeuAcHex4HexNAc3dHex3 2003 −−− −−− −−− −−− NeuAcHex5HexNAc4SP 2010 −−−−−− −−− Hex5HexNAc4dHex2SP 2011 −−− −−− ++ NeuAc2Hex5HexNAc3 2018 +++NeuAcHex5HexNAc3dHex2 2019 +++ Hex6HexNAc4dHexSP 2027 ++ + ++NeuAcHex6HexNAc3dHex 2035 −−− + −−− −−−NeuAc2Hex3HexNAc4dHex/Hex7HexNAc4SP 2043 +++ +++ NeuAcHex7HexNAc3 2051 −−−− Hex4HexNAc5dHex2SP 2052 −−− −−− ++ Hex5HexNAc5dHexSP 2068 +++ ++++++ NeuAcHex5HexNAc4dHex 2076 + −− NeuGcHex5HexNAc4dHex/NeuAcHex6HexNAc42092 − − − NeuGcHex6HexNAc4 2108 − + NeuAcHex4HexNAc5dHex 2117 + + −NeuAcHex5HexNAc5 2133 + ++ NeuAcHex5HexNAc4dHexSP/ 2156 + −−−NeuAcHex8HexNAc2dHex Hex5HexNAc4dHex3SP 2157 +++ +++NeuAc2Hex5HexNAc3dHex 2164 −−− NeuAcHex5HexNAc3dHex3 2165 +++NeuAcHex9HexNAc2/NeuAcHex6HexNAc4SP/ 2172 +++ NeuGcHex5HexNAc4dHexSPNeuAcHex4HexNAc6 2174 −−− −−− −−− −−−NeuAc2Hex3HexNAc4dHex2/Hex7HexNAc4dHexSP 2189 −−− NeuAcHex3HexNAc4dHex42190 −−− −−− −−− ++ NeuGcNeuAcHex6HexNAc3/ 2196 +++ +++NeuGc2Hex5HexNAc3dHex Hex4HexNAc5dHexSP 2198 −−− −−− −−−NeuAc2Hex4HexNAc4(SP)2 2219 +++ NeuAc2Hex5HexNAc4 2221 −− −−NeuAcHex5HexNAc4dHex2 2222 − −− −−−?? −− Hex6HexNAc5dHexSP 2230 ++ −−−−−− ++ NeuGcNeuAcHex5HexNAc4 2237 +++ +++NeuGcHex5HexNAc4dHex2/NeuAcHex6HexNAc4dHex 2238 −− − − −−NeuGc2Hex5HexNAc4 2253 + ++ −−− −−−NeuAcHex7HexNAc4/NeuGcHex6HexNAc4dHex 2254 ++ − ++ ++NeuAcHex4HexNAc5dHex2 2263 −−− −−− −−− NeuAcHex5HexNAc5dHex 2279 + + −NeuAcHex6HexNAc5 2295 + NeuAcHex5HexNAc3dHex4/NeuGcHex6HexNAc5 2311 ++++++ Hex6HexNAc4dHex3SP 2319 −−− −−− ++ −−− NeuAc2Hex5HexNAc4dHex 2367 −−− −−− NeuAcHex5HexNAc4dHex3 2368 −−− − −−− −−−NeuGcNeuAcHex5HexNAc4dHex/ 2383 −− − −−− NeuAc2Hex6HexNAc4NeuGcHex5HexNAc4dHex3/NeuAcHex6HexNAc4dHex2 2384 +++NeuAc3Hex5HexNAx3SP/NeuAc2Hex5HexNAc4Ac4 2389 −−− + + −−−NeuAc2Hex5HexNAc3dHexSP 2390 +++ NeuAc2Hex3HexNAc5dHex2 2392 +++NeuAcHex3HexNAc5dHex4 2393 +++ NeuGc2Hex5HexNAc4dHex 2399 −−− −−− −−−−−− NeuAc2Hex6HexNAc3dHexSP 2406 −−− ++ −−− −−− NeuAc2Hex4HexNAc5dHex2408 −−− −−− −−− −−− NeuAcHex5HexNAc5dHex2 2425 +++ NeuAcHex6HexNAc5dHex2441 + + + NeuAc2Hex5HexNAc4dHexSP/ 2447 −−− −−− −−− −−−NeuAc2Hex8HexNAc2dHex NeuAcHex5HexNAc4dHex3SP/ 2448 −−− −−− −−− −−−NeuAcHex8HexNAc2dHex3 NeuAcHex3HexNAc6dHex3 2450 +++ NeuAcHex7HexNAc52457 ++ NeuAc3Hex5HexNAc4 2512 −−− −−− −−− NeuAc2Hex5HexNAc4dHex2 2513−−− −−− −−− −−− NeuAcHex6HexNAc5dHexSP 2521 +++ NeuGcNeuAc2Hex5HexNAc42528 −−− −−− −−− NeuGcNeuAcHex5HexNAc4dHex2/ 2529 −−− −−− −−− −−−NeuAc2Hex6HexNAc4dHex NeuGc2NeuAcHex5HexNAc4 2544 −−− −−− −−− −−−NeuAc2Hex6HexNAc5 2586 −−− + −−− −−− NeuAcHex6HexNAc5dHex2 2587 −−− −−−Hex7HexNAc6dHexSP 2595 +++ +++NeuAcHex7HexNAc5dHex/NeuGcHex6HexNAc5dHex2 2603 +NeuAcHex8HexNAc5/NeuGcHex7HexNAc5dHex 2619 −−− NeuAcHex6HexNAc6dHex 2644+++ NeuAcHex7HexNAc6 2660 −−− −−− + NeuAc2Hex6HexNAc5dHex 2732 − −−−NeuAcHex6HexNAc5dHex3 2733 −−− −−− −−− NeuAc2Hex4HexNAc6dHex2 2758 ++++++ NeuAcHex8HexNAc5dHex 2765 − −− NeuGcHex8HexNAc5dHex/NeuAcHex9HexNAc52781 −−− −−− NeuAc2Hex5HexNAc4dHex4 2806 ++ +++ NeuAcHex7HexNAc6dHex2807 +++ +++ −−− NeuAcHex8HexNAc6 2822 +++ +++ NeuAc3Hex6HexNAc5 2878−−− −−− −−− −−− NeuGcNeuAc2Hex6HexNAc5 2894 −−− −−− −−− −−−NeuGcNeuAcHex6HexNAc5dHex2/ 2895 +++ NeuAc2Hex7HexNAc5dHexNeuAc2Hex7HexNAc6 2952 −−− −−− −−− NeuAcHex7HexNAc6dHex2 2953 +++NeuAc3Hex6HexNAc5dHex 3024 −−− + −−− −−− NeuAc2Hex7HexNAc6dHex 3098 −−−−−− −−− −−− NeuAcHex8HexNAc7dHex 3172 +++ ¹⁾Code: +++ new signalappeared, ++ highly increased relative signal intensity, ++ increasedrelative signal intensity, − decreased relative signal intensity, −−greatly decreased relative signal intensity, −−− signal disappeared,blank: no change.

TABLE 26 Preferred monosaccharide Terminal Experimental structuresincluded in the glycan m/z* compositions epitopes signal according tothe invention^(§) Group^(#) 730 Hex3HexNAc Manα (Manα→)₂Hex₁HexNAc₁ S771 Hex2HexNAc2 Manα Manα→Hex₁HexNAc₂ LO 892 Hex4HexNAc Manα(Manα→)₃Hex₁HexNAc₁ S Galβ4 Galβ4GlcNAc→Hex₃ 917 Hex2HexNAc2dHex ManαManα→Hex₁HexNAc₂dHex₁ LO, F 933 Hex3HexNAc2 Manα (Manα→)₂Hex₁HexNAc₂ LO1054 Hex5HexNAc Manα (Manα→)₄Hex₁HexNAc₁ S 1079 Hex3HexNAc2dHex Manα(Manα→)₂Hex₁HexNAc₂dHex₁ LO, F 1095 Hex4HexNAc2 Manα (Manα→)₃Hex₁HexNAc₂LO 1120 Hex2HexNAc3dHex Fucα3/4 Fucα3/4→Hex₂HexNAc₃ HY, F, N > H 1136Hex3HexNAc3 GlcNAcβ GlcNAcβ→Hex₃HexNAc₂ HY, N═H 1216 Hex6HexNAc Manα(Manα→)₅Hex₁HexNAc₁ S 1241 Hex4HexNAc2dHex Manα (Manα)₃Hex₁HexNAc₂dHex₁LO, F 1257 Hex5HexNAc2 Manα (Manα→)₄Hex₁HexNAc₂ HI 1282 Hex3HexNAc3dHexGlcNAcβ GlcNAcβ→Hex₃HexNAc₂dHex₁ HY, F, N═H 1298 Hex4HexNAc3 HY 1339Hex3HexNAc4 2 × GlcNAcβ (GlcNAcβ→)₂Hex₃HexNAc₂ CO, N > H 1378 Hex7HexNAcManα (Manα→)₆Hex₁HexNAc₁ S 1403 Hex5HexNAc2dHex Manα(Manα→)₄Hex₁HexNAc₂dHex₁ HF 1419 Hex6HexNAc2 Manα (Manα→)₅Hex₁HexNAc₂ HI1444 Hex4HexNAc3dHex Manα Manα→Hex₃HexNAc₃dHex₁ HY, F 1460 Hex5HexNAc3Manα Manα→Hex₄HexNAc₃ HY 1485 Hex3HexNAc4dHex 2 × GlcNAcβ(GlcNAcβ→)₂Hex₃HexNAc₂dHex₁ CO, F, N > H 1501 Hex4HexNAc4 GlcNAcβGlcNAcβ→Hex₄HexNAc₃ CO, Galβ4 Galβ4GlcNAc→Hex₃HexNAc₃ N═H 1540Hex8HexNAc Manα (Manα→)₇Hex₁HexNAc₁ S 1542 Hex3HexNAc5 3 × GlcNAcβ(GlcNAcβ→)₃Hex₃HexNAc₂ CO, N > H 1565 Hex6HexNAc2dHex Manα(Manα→)₅Hex₁HexNAc₂dHex₁ HF 1581 Hex7HexNAc2 Manα (Manα→)₆Hex₁HexNAc₂ HI1590 Hex4HexNAc3dHex2 Fucα Fucα→Hex₄HexNAc₃dHex₁ HY, FC 1606Hex5HexNAc3dHex Manα Manα→Hex₄HexNAc₃dHex₁ HY, F Galβ4Galβ4GlcNAc→Hex₄HexNAc₂dHex₁ Manα→[Galβ4GlcNAc→]Hex₃HexNAc₂dHex₁ 1622Hex6HexNAc3 Manα Manα→Hex₅HexNAc₃ HY Galβ4 Galβ4GlcNAc→Hex₅HexNAc₂Manα→[Galβ4GlcNAc→]Hex₄HexNAc₂ 1647 Hex4HexNAc4dHex GlcNAcβGlcNAcβ→Hex₄HexNAc₃dHex₁ CO, F, Galβ4 Galβ4GlcNAc→Hex₃HexNAc₃dHex₁ N═HGlcNAcβ→[Galβ4GlcNAc→]Hex₃HexNAc₂dHex₁ 1663 Hex5HexNAc4 2 × Galβ4(Galβ4GlcNAc→)₂Hex₃HexNAc₂ CO 1688 Hex3HexNAc5dHex 3 × GlcNAcβ(GlcNAcβ→)₃Hex₃HexNAc₂dHex₁ CO, F, Manα Manα→Hex₂HexNAc₅dHex₁ N > H 1702Hex9HexNAc Manα (Manα→)₈Hex₁HexNAc₁ S 1704 Hex4HexNAc5 2 × HexNAcβHexNAcβHexNAcβ→Hex₄HexNAc₃dHex₁ CO, (not Galβ4GlcNAc→Hex₃HexNAc₄dHex₁N > H GlcNAc) HexNAcβHexNAcβ→[Galβ4GlcNAc→] Galβ4 Hex₃HexNAc₂dHex₁ 1743Hex8HexNAc2 Manα (Manα→)₇Hex₁HexNAc₂ HI 1768 Hex6HexNAc3dHex ManαManα→Hex₅HexNAc₃dHex₁ HY, F 1784 Hex7HexNAc3 Manα Manα→Hex₆HexNAc₃ HYGalβ4 Galβ4GlcNAc→Hex₆HexNAc₂ Manα→[Galβ4GlcNAc→]Hex₅HexNAc₂ 1793Hex4HexNAc4dHex2 GlcNAcβ GlcNAcβ→Hex₄HexNAc₃dHex₂ CO, FC, Galβ4Galβ4GlcNAc→Hex₃HexNAc₃dHex₂ N═H Fucα3/4 Fucα3/4→Hex₄HexNAc₄dHex₁GlcNAcβ→[Galβ4GlcNAc→]Hex₃HexNAc₂dHex₂GlcNAcβ→[Fucα3/4→]Hex₄HexNAc₃dHex₁Fucα3/4→[Galβ4GlcNAc→]Hex₃HexNAc₃dHex₁ GlcNAcβ→[Fucα3/4→][Galβ4GlcNAc→]Hex₄HexNAc₃dHex₁ 1809 Hex5HexNAc4dHex 2 × Galβ4(Galβ4GlcNAc→)₂Hex₃HexNAc₂dHex₁ CO, F 1850 Hex4HexNAc5dHex 2 × GlcNAcβ(GlcNAcβ→)₂Hex₄HexNAc₃dHex₁ CO, F, Galβ4 Galβ4GlcNAc→Hex₃HexNAc₄dHex₁N > H Galβ4GlcNAc→[GlcNAcβ→]₂Hex₃HexNAc₂dHex₁ 1866 Hex5HexNAc5 CO, N═H1905 Hex9HexNAc2 Manα (Manα→)₈Hex₁HexNAc₂ HI 1955 Hex5HexNAc4dHex2Fucα3/4 Fucα3/4→Hex₅HexNAc₄dHex₁ CO, FC Galβ4Galβ4GlcNAc→Hex₄HexNAc₃dHex₂ Galβ4GlcNAc→[Fucα3/4→]Hex₄HexNAc₃dHex₁ 1971Hex6HexNAc4dHex Galβ4 Galβ4GlcNAc→Hex₅HexNAc₃dHex₁ CO, F 1996Hex4HexNAc5dHex2 2 × GlcNAcβ (GlcNAcβ→)₂Hex₄HexNAc₃dHex₂ CO, FC, Fucα3/4Fucα3/4→Hex₄HexNAc₅dHex₁ N > H Galβ4 Galβ4GlcNAc→Hex₃HexNAc₄dHex₂(GlcNAcβ→)₂[Fucα3/4→]Hex₄HexNAc₃dHex₁Galβ4GlcNAc→[Fucα3/4→]Hex₃HexNAc₄dHex₁ 2012 Hex5HexNAc5dHex GlcNAcβGlcNAcβ→Hex₅HexNAc₄dHex₁ CO, F, N═H 2028 Hex6HexNAc5 Galβ4Galβ4GlcNAc→Hex₅HexNAc₄ CO 3 × Galβ4 (Galβ4GlcNAc→)₃Hex₃HexNAc₂ 2067Hex10HexNAc2 Manα Glc→(Manα→)₈Hex₁HexNAc₂ G Glc 2101 Hex5HexNAc4dHex3GlcNAcβ GlcNAcβ→Hex₅HexNAc₃dHex₃ CO, FC 2174 Hex6HexNAc5dHex 3 × Galβ4(Galβ4GlcNAc→)₃Hex₃HexNAc₂dHex₁ CO, F 2229 Hex11HexNAc2 ManαGlc₂→(Manα→)₈Hex₁HexNAc₂ G Glc 2320 Hex6HexNAc5dHex2 Galβ4Galβ4GlcNAc→Hex₅HexNAc₄dHex₂ CO, FC 2391 Hex12HexNAc2 ManαGlc₃→(Manα→)₈Hex₁HexNAc₂ G Glc *[M + Na]⁺ ion, first isotope. ^(§)“→”indicates linkage to a monosaccharide in the rest of the structure; “[]” indicates branch in the structure. ^(#)Preferred structure groupbased on monosaccharide compositions according to the present invention.HI, high-mannose; LO, low-mannose; S, soluble mannosylated; HF,fucosylated high-mannose; G, glucosylated high-mannose; HY, hybrid-typeor monoantennary; CO, complex-type; F, fucosylation; FC, complexfucosylation; N═H, terminal HexNAc (HexNAc═Hex); N > H, terminal HexNAc(HexNAc > Hex).

TABLE 27 Preferred Terminal m/z* monosaccharide compositions epitopesGroup^(#) 989 Hex3HexNAc2SP SP 1030 Hex2HexNAc3SP HY, SP, N > H 1151Hex4HexNac2SP SP 1192 Hex3HexNAc3SP HY, SP 1272 NeuAc2Hex2HexNAcdHexNeuAcα6/8/9 F Fucα3/4 1297 Hex4HexNAc2dHexSP F, SP 1313NeuAc2HexHexNAc2dHex Fucα2 F 1338 Hex3HexNAc3dHexSP Fucα3/4 HY, F, SP1354 Hex4HexNAc3SP HY, SP 1395 Hex3HexNac4SP CO, SP, N > H 1403NeuAcHex3HexNAc3 NeuAcα6/8/9 HY 1419 NeuGcHex3HexNAc3 HY 1475NeuAc2Hex2HexNAcdHex F 1500 Hex4HexNAc3dHexSP HY, F, SP 1516Hex5HexNAc3dHexSP/ HY, F NeuAc2HexHexNAc3dHex (SP) 1541Hex3HexNAc4dHexSP CO, F, SP, N > H 1549 NeuAcHex3HexNAc3dHex NeuAcα6/8/9HY, F 1557 Hex4HexNAc4SP CO, SP 1565 NeuAcHex4HexNAc3 NeuAcα6/8/9 HYNeuAcα3 1581 NeuGcHex4HexNAc3 HY 1637 NeuAc2Hex3HexNAc2dHex F 1662Hex5HexNAc3dHexSP Fucα3/4 HY, F, SP 1678 NeuAc2Hex2HexNAc3dHex Fucα3/4HY, F, N > H 1703 Hex4HexNAc4dHexSP CO, F, SP 1711 NeuAcHex4HexNAc3dHexNeuAcα6/8/9 HY, F 1719 Hex5HexNAc4SP CO, SP 1727 NeuAcHex5HexNAc3NeuAcα6/8/9 HY NeuAcα3 Fucα3/4 1743 NeuGcHex5HexNAc3 NeuGcα3 HY 1752NeuAcHex3HexNAc4dHex NeuAcα6/8/9 CO, F, Fucα2 N > H 1760 Hex4HexNAc5SPCO, SP, N > H 1768 NeuAcHex4HexNAc4 NeuAcα6/8/9 CO 1783Hex7HexNAc2dHexSP F, SP 1799 Hex5HexNAc4SP2/ (CO) NeuAc2Hex4HexNAc2dHex(F) (SP) 1840 NeuAc2Hex3HexNAc3dHex HY, F 1865 Hex5HexNAc4dHexSP CO, F,SP 1873 NeuAcHex5HexNAc3dHex NeuAcα6/8/9 HY, F NeuAcα3 Fucα2 1881Hex6HexNAc4SP CO, SP 1889 NeuAcHex6HexNAc3 NeuAcα6/8/9 HY NeuAcα3 1906Hex4HexNAc5dHexSP CO, F, SP, N > H 1914 NeuAcHex4HexNAc4dHex NeuAcα6/8/9CO, F NeuAcα3 1930 NeuAcHex5HexNAc4 NeuAcα6/8/9 CO 1946 NeuGcHex5HexNAc4CO 1955 NeuAcHex3HexNAc5dHex NeuAcα6/8/9 CO, F, Fucα2 N > H 1971NeuAcHex4HexNAc5 CO, N > H 2002 NeuAc2Hex4HexNAc3dHex/ Fucα2 HY (F)Hex8HexNAc3SP (SP) 2003 NeuAcHex4HexNAc3dHex3 NeuAcα3 HY, FC NeuAcα6/8/9Fucα3/4 2010 NeuAcHex5HexNAc4SP NeuAcα6/8/9 CO, SP Fucα3/4 2011Hex5HexNAc4dHex2SP NeuAcα3 CO, FC, Fucα2 SP 2027 Hex6HexNAc4dHexSP CO,F, SP 2035 NeuAcHex6HexNAc3dHex NeuAcα3 HY, F NeuAcα6/8/9 Fucα2 2051NeuAcHex7HexNAc3 NeuAcα6/8/9 HY Fucα3/4 2052 Hex4HexNAc5dHex2SP NeuAcα3SP Fucα2 2076 NeuAcHex5HexNAc4dHex NeuAcα6/8/9 CO, F 2092NeuGcHex5HexNAc4dHex/ NeuAcα3 CO (F) NeuAcHex6HexNAc4 Fucα3/4 2108NeuGcHex6HexNAc4 NeuGcα3 CO 2117 NeuAcHex4HexNAc5dHex NeuAcα6/8/9 CO, F2133 NeuAcHex5HexNAc5 CO, N═H 2156 NeuAcHex5HexNAc4dHexSP/ NeuAcα6/8/9(CO) F NeuAcHex8HexNAc2dHex (SP) 2164 NeuAc2Hex5HexNAc3dHex Fucα2 HY, F2174 NeuAcHex4HexNAc6 NeuAcα3 CO, NeuAcα6/8/9 N > H Fucα3/4 2189NeuAc2Hex3HexNAc4dHex2/ Fucα2 CO Hex7HexNAc4dHexSP F(C) (SP) (N > H)2190 NeuAcHex3HexNAc4dHex4 NeuAcα3 CO, FC, Fucα3/4 N > H 2198Hex4HexNAc5dHexSP NeuAcα3 CO, F, Fucα3/4 SP, N > H 2221NeuAc2Hex5HexNAc4 NeuAcα3 CO NeuAcα6/8/9 2222 NeuAcHex5HexNAc4dHex2NeuAcα3 CO, FC NeuAcα6/8/9 Fucα3/4 Fucα2 2230 Hex6HexNAc5dHexSP Fucα3/4CO, F, SP 2238 NeuGcHex5HexNAc4dHex2/ NeuAcα3 CO, NeuAcHex6HexNAc4dHexNeuAcα6/8/9 F(C) Fucα3/4 2253 NeuGc2Hex5HexNAc4 NeuAcα6/8/9 CO Fucα22254 NeuAcHex7HexNAc4/ Fucα3/4 CO (F) NeuGcHex6HexNAc4dHex 2263NeuAcHex4HexNAc5dHex2 NeuAcα6/8/9 CO, FC, Fucα3/4 N > H 2279NeuAcHex5HexNAc5dHex NeuAcα6/8/9 CO, F, N═H 2295 NeuAcHex6HexNAc5 CO2319 Hex6HexNAc4dHex3SP NeuAcα3 CO, FC, NeuAcα6/8/9 SP Fucα3/4 2367NeuAc2Hex5HexNAc4dHex NeuAcα6/8/9 CO, F NeuAcα3 Fucα2 2368NeuAcHex5HexNAc4dHex3 NeuAcα3 CO, FC NeuAcα6/8/9 Fucα2 Fucα3/4 2383NeuGcNeuAcHex5HexNAc4dHex/ NeuAcα6/8/9 CO (F) NeuAc2Hex6HexNAc4 NeuAcα3Fucα2 2389 NeuAc3Hex5HexNAc3SP NeuAcα3 HY, SP NeuAcα6/8/9 2399NeuGc2Hex5HexNAc4dHex NeuAcα3 CO, F NeuAcα6/8/9 Fucα3/4 2406NeuAc2Hex6HexNAc3dHexSP NeuAcα3 HY, F, NeuAcα6/8/9 SP Fucα2 2408NeuAc2Hex4HexNAc5dHex NeuAcα3 CO, F, NeuAcα6/8/9 N > H Fucα3/4 2441NeuAcHex6HexNAc5dHex CO, F 2447 NeuAc2Hex5HexNAc4dHexSP NeuAcα3 CO, F,NeuAcα6/8/9 SP Fucα3/4 2448 NeuAcHex5HexNAc4dHex3SP NeuAcα3 CO, FC,NeuAcα6/8/9 SP Fucα3/4 2457 NeuAcHex7HexNAc5 CO 2512 NeuAc3Hex5HexNAc4NeuAcα3 CO NeuAcα6/8/9 Fucα2 2513 NeuAc2Hex5HexNAc4dHex2 NeuAcα3 CO, FCNeuAcα6/8/9 Fucα3/4 2528 NeuGcNeuAc2Hex5HexNAc4 NeuAcα3 CO NeuAcα6/8/9Fucα2 2529 NeuGcNeuAcHex5HexNAc4dHex2/ NeuAcα3 CO, NeuAc2Hex6HexNAc4dHexNeuAcα6/8/9 F(C) Fucα3/4 2544 NeuGc2NeuAcHex5HexNAc4 NeuAcα3 CONeuAcα6/8/9 Fucα3/4 2586 NeuAc2Hex6HexNAc5 NeuAcα3 CO NeuAcα6/8/9 Fucα22587 NeuAcHex6HexNAc5dHex2 NeuAcα3 CO, FC NeuAcα6/8/9 2603NeuAcHex7HexNAc5dHex/ CO, NeuGcHex6HexNAc5dHex2 F(C) 2619NeuAcHex8HexNAc5/ Fucα2 CO (F) NeuGcHex7HexNAc5dHex 2660NeuAcHex7HexNAc6 Fucα3/4 CO 2732 NeuAc2Hex6HexNAc5dHex NeuAcα6/8/9 CO, FNeuAcα3 2733 NeuAcHex6HexNAc5dHex3 NeuAcα3 CO, FC NeuAcα6/8/9 Fucα2 2765NeuAcHex8HexNAc5dHex NeuAcα6/8/9 CO, F NeuAcα3 2781NeuGcHex8HexNAc5dHex/ Fucα3/4 CO (F) NeuAcHex9HexNAc5 2878NeuAc3Hex6HexNAc5 NeuAcα3 CO NeuAcα6/8/9 Fucα3/4 2894NeuGcNeuAc2Hex6HexNAc5 NeuAcα3 CO NeuAcα6/8/9 Fucα3/4 2952NeuAc2Hex7HexNAc6 NeuAcα6/8/9 CO 3024 NeuAc3Hex6HexNAc5dHex NeuAcα3 CO,F NeuAcα6/8/9 Fucα2 3098 NeuAc2Hex7HexNAc6dHex NeuAcα3 CO, F NeuAcα6/8/9Fucα3/4 *[M − H]⁻ ion, first isotope. ^(#)Preferred structure groupbased on monosaccharide compositions according to the present invention.HY, hybrid-type or monoantennary; CO, complex-type; F, fucosylation; FC,complex fucosylation; N═H, terminal HexNAc (HexNAc═Hex); N > H, terminalHexNAc (HexNAc > Hex); SP, sulphate and/or phosphate ester; “( )”indicates that the glycan signal includes also other structure types.

TABLE 28 Preferred monosaccharide Terminal Experimental structuresincluded in the glycan m/z* compositions epitopes signal according tothe invention^(§) Group^(#) 1825 Hex6HexNAc4 Galβ4Galβ4GlcNAc→Hex₅HexNAc₃ CO Galα Galα3Gal→Hex₄HexNAc₄Galβ4GlcNAc→[Galα3Gal→]Hex₃HexNAc₃ 1987 Hex7HexNAc4 GalαGalα3Gal→Hex₅HexNAc₄ CO (Galα3Gal→)₂Hex₃HexNAc₄ 2133 Hex7HexNAc4dHex1Galα Galα3Gal→Hex₅HexNAc₄dHex₁ CO, F (Galα3Gal→)₂Hex₃HexNAc₄dHex₁ 2190Hex7HexNAc5 Galα Galα3Gal→Hex₅HexNAc₅ CO 2336 Hex7HexNAc5dHex Galβ4Galβ4GlcNAc→Hex₆HexNAc₄dHex₁ CO, F Galα Galα3Gal→Hex₅HexNAc₅dHex₁Galβ4GlcNAc→[Galα3Gal→]Hex₄HexNAc₄dHex₁ 2352 Hex8HexNAc5 Galβ4Galβ4GlcNAc→Hex₇HexNAc₄ CO Galα Galα3Gal→Hex₆HexNAc₅Galβ4GlcNAc→[Galα3Gal→]Hex₅HexNAc₄ Galβ4GlcNAc→[Galα3Gal→]₂Hex₃HexNAc₄2498 Hex8HexNAc5dHex Galβ4 Galβ4GlcNAc→Hex₇HexNAc₄dHex₁ CO, F GalαGalα3Gal→Hex₆HexNAc₅dHex₁ Galβ4GlcNAc→[Galα3Gal→]Hex₅HexNAc₄dHex₁Galβ4GlcNAc→[Galα3Gal→]₂Hex₃HexNAc₄dHex₁ 2514 Hex9HexNAc5 GalαGalα3Gal→Hex₇HexNAc₅ CO (Galα3Gal→)₂Hex₅HexNAc₅ (Galα3Gal→)₃Hex₃HexNAc₅2660 Hex9HexNAc5dHex Galα Galα3Gal→Hex₇HexNAc₅dHex₁ CO, F(Galα3Gal→)₂Hex₅HexNAc₅dHex₁ (Galα3Gal→)₃Hex₃HexNAc₅dHex₁ *[M + Na]⁺ion, first isotope. ^(§)“→” indicates linkage to a monosaccharide in therest of the structure; “[ ]” indicates branch in the structure.^(#)Preferred structure group based on monosaccharide compositionsaccording to the present invention. HI, high-mannose; LO, low-mannose;S, soluble mannosylated; HF, fucosylated high-mannose; G, glucosylatedhigh-mannose; HY, hybrid-type or monoantennary; CO, complex-type; F,fucosylation; FC, complex fucosylation; N═H, terminal HexNAc (HexNAc =Hex); N > H, terminal HexNAc (HexNAc > Hex).

TABLE 29 Proposed composition m/z α-Man β-GlcNAc β4-Gal β3-GalHex2HexNAc 568 −−− HexHexNAc2 609 +++ Hex2HexNAcdHex 714 +++ Hex3HexNAc730 −− −−− HexHexNAc2dHex 755 +++ Hex2HexNAc2 771 ++ ++ Hex4HexNAc 892−−− + Hex2HexNAc2dHex 917 + Hex3HexNAc2 933 ++ ++ Hex2HexNAc3 974 +++Hex5HexNAc 1054 −− Hex3HexNAc2dHex 1079 −− + Hex4HexNAc2 1095 − +Hex2HexNAc3dHex 1120 +++ −−− Hex3HexNAc3 1136 ++ −− + Hex2HexNAc2dHex31209 −−− −−− Hex6HexNAc 1216 −− Hex4HexNAc2dHex 1241 −−− Hex5HexNAc21257 −− Hex2HexNAc3dHex2 1266 Hex3HexNAc3dHex 1282 ++ −− + Hex4HexNAc31298 ++ − Hex3HexNAc4 1339 +++ +++ Hex7HexNAc 1378 −− Hex5HexNAc2dHex1403 −−− Hex6HexNAc2 1419 −− + Hex3HexNAc3dHex2 1428 +++ Hex4HexNAc3dHex1444 + − + Hex5HexNAc3 1460 + − ++ Hex3HexNAc4dHex 1485 −− ++Hex4HexNAc4 1501 ++ Hex8HexNAc 1540 − Hex3HexNAc5 1542 +++Hex6HexNAc2dHex 1565 −−− −−− −−− Hex7HexNAc2 1581 −− Hex4HexNAc3dHex21590 Hex5HexNAc3dHex 1606 −− −− Hex6HexNAc3 1622 −− − −− Hex4HexNAc4dHex1647 −−− Hex5HexNAc4 1663 −− −−− Hex3HexNAc5dHex 1688 −−− ++ Hex9HexNAc1702 −−− −−− Hex8HexNAc2 1743 −− + Hex6HexNAc3dHex 1768 −−− Hex7HexNAc31784 −−− −−− −−− Hex4HexNAc4dHex2 1793 −−− ++ Hex5HexNAc4dHex 1809 −−−−− Hex3HexNAc6dHex 1891 +++ Hex9HexNAc2 1905 −−− − Hex5HexNAc4dHex21955 − −−− Hex6HexNAc4dHex 1971 −−− −−− Hex4HexNAc5dHex2 1996 −−−Hex5HexNAc5dHex 2012 −−− −−− −−− Hex6HexNAc5 2028 − −−− Hex10HexNAc22067 −−− − Hex5HexNAc4dHex3 2101 −−− Hex4HexNAc5dHex3 2142 −− −−−Hex6HexNAc5dHex 2174 −− −−− Hex11HexNAc2 2229 Hex5HexNAc5dHex3 2304 −−−Hex6HexNAc5dHex2 2320 −−− Hex7HexNAc6 2393 −−− Hex6HexNAc5dHex3 2466 −−−Hex7HexNAc6dHex 2539 −−− −−−

TABLE 30 Preferred monosaccharide Terminal Experimental structuresincluded in the glycan m/z* compositions epitopes signal according tothe invention^(§) Group^(#) 568 Hex2HexNAc Manα Manα→Hex₁HexNAc₁ S 730Hex3HexNAc Manα (Manα→)₂Hex₁HexNAc₁ S GlcNAc GlcNAc→Hex₃ 771 Hex2HexNAc2Manα Manα→Hex₁HexNAc₂ LO 892 Hex4HexNAc Manα (Manα→)₃Hex₁HexNAc₁ S 917Hex2HexNAc2dHex Manα Manα→Hex₁HexNAc₂dHex₁ LO, F 933 Hex3HexNAc2 Manα(Manα→)₂Hex₁HexNAc₂ LO 1054 Hex5HexNAc Manα (Manα→)₄Hex₁HexNAc₁ S 1079Hex3HexNAc2dHex Manα (Manα→)₂Hex₁HexNAc₂dHex₁ LO, F 1095 Hex4HexNAc2Manα (Manα→)₃Hex₁HexNAc₂ LO 1120 Hex2HexNAc3dHex GlcNAcβGlcNAcβ→Hex₂HexNAc₂dHex₁ HY, F, N > H 1136 Hex3HexNAc3 GlcNAcβGlcNAcβ→Hex₃HexNAc₂ HY, N═H 1209 Hex2HexNAc2dHex3 ManαManα→Hex₁HexNAc₂dHex₃ FC, GlcNAc GlcNAc→Hex₂HexNAc₁dHex₃ N═H 1216Hex6HexNAc Manα (Manα→)₅Hex₁HexNAc₁ S 1241 Hex4HexNAc2dHex Manα(Manα)₃Hex₁HexNAc₂dHex₁ LO, F 1257 Hex5HexNAc2 Manα (Manα→)₄Hex₁HexNAc₂HI 1266 Hex2HexNAc3dHex2 Fuc Fuc→Hex₂HexNAc₃dHex₁ HY, FC 1282Hex3HexNAc3dHex GlcNAcβ GlcNAcβ→Hex₃HexNAc₂dHex₁ HY, F, N═H 1298Hex4HexNAc3 HY 1378 Hex7HexNAc Manα (Manα→)₆Hex₁HexNAc₁ S 1403Hex5HexNAc2dHex Manα (Manα)₄Hex₁HexNAc₂dHex₁ HF 1419 Hex6HexNAc2 Manα(Manα→)₅Hex₁HexNAc₂ HI 1444 Hex4HexNAc3dHex GlcNAcβGlcNAcβ→Hex₄HexNAc₂dHex₁ HY, F 1460 Hex5HexNAc3 GlcNAcβGlcNAcβ→Hex₅HexNAc₂ HY 1485 Hex3HexNAc4dHex 2 × GlcNAcβ(GlcNAcβ→)₂Hex₃HexNAc₂dHex₁ CO, F, N > H 1501 Hex4HexNAc4 CO, N═H 1540Hex8HexNAc Manα (Manα→)₇Hex₁HexNAc₁ S 1565 Hex6HexNAc2dHex Manα(Manα)₅Hex₁HexNAc₂dHex₁ HF 1581 Hex7HexNAc2 Manα (Manα→)₆Hex₁HexNAc₂ HI1590 Hex4HexNAc3dHex2 Fucα Fucα→Hex₄HexNAc₃dHex₁ HY, FC 1606Hex5HexNAc3dHex GlcNAcβ GlcNAcβ→Hex₅HexNAc₂dHex₁ HY, F Galβ4Galβ4GlcNAc→Hex₄HexNAc₂dHex₁ 1622 Hex6HexNAc3 Manα Manα→Hex₅HexNAc₃ HYGlcNAcβ GlcNAcβ→Hex₆HexNAc₂ Galβ4 Galβ4GlcNAc→Hex₅HexNAc₂Manα→[GlcNAcβ→]Hex₅HexNAc₂ Manα→[Galβ4GlcNAc→]Hex₄HexNAc₂ 1647Hex4HexNAc4dHex GlcNAcβ GlcNAcβ→Hex₄HexNAc₃dHex₁ CO, F, N═H 1663Hex5HexNAc4 2 × Galβ4 (Galβ4GlcNAc→)₂Hex₃HexNAc₂ CO GlcNAcβGlcNAcβ→Hex₅HexNAc₃ 1688 Hex3HexNAc5dHex 3 × GlcNAcβ(GlcNAcβ→)₃Hex₃HexNAc₂dHex₁ CO, F, N > H 1702 Hex9HexNAc Manα(Manα→)₈Hex₁HexNAc₁ S 1743 Hex8HexNAc2 Manα (Manα→)₇Hex₁HexNAc₂ HI 1768Hex6HexNAc3dHex Galβ4 Galβ4GlcNAc→Hex₅HexNAc₂dHex₁ HY, F 1784Hex7HexNAc3 Manα Manα→Hex₆HexNAc₃ HY GlcNAcβ GlcNAcβ→Hex₇HexNAc₂ Galβ4Galβ4GlcNAc→Hex₆HexNAc₂ Manα→[GlcNAcβ→]Hex₆HexNAc₂Manα→[Galβ4GlcNAc→]Hex₅HexNAc₂ 1793 Hex4HexNAc4dHex2 GlcNAcβGlcNAcβ→Hex₄HexNAc₃dHex₂ CO, FC, Fuc Fuc→Hex₄HexNAc₄dHex₁ N═HGlcNAcβ→[Fuc→]Hex₄HexNAc₃dHex₁ 1809 Hex5HexNAc4dHex 2 × Galβ4(Galβ4GlcNAc→)₂Hex₃HexNAc₂dHex₁ CO, F GlcNAcβ GlcNAcβ→Hex₅HexNAc₃dHex₁1891 Hex3HexNAc6dHex CO, F, N > H 1905 Hex9HexNAc2 Manα(Manα→)₈Hex₁HexNAc₂ HI 1955 Hex5HexNAc4dHex2 Galβ4Galβ4GlcNAc→Hex₄HexNAc₃dHex₂ CO, FC Fuc Fuc→Hex₅HexNAc₄dHex₁Galβ4GlcNAc→[Fuc→]Hex₄HexNAc₃dHex₁ 1971 Hex6HexNAc4dHex GlcNAcβGlcNAcβ→Hex₆HexNAc₃dHex₁ CO, F Galβ4 Galβ4GlcNAc→Hex₅HexNAc₃dHex₁ 1996Hex4HexNAc5dHex2 2 × GlcNAcβ (GlcNAcβ→)₂Hex₄HexNAc₃dHex₂ CO, FC, N > H2012 Hex5HexNAc5dHex GlcNAcβ GlcNAcβ→Hex₅HexNAc₄dHex₁ CO, F, 2 × Galβ4(Galβ4GlcNAc→)₂Hex₃HexNAc₃dHex₁ N═H Galβ3 Galβ3GlcNAc→Hex₄HexNAc₄dHex₁(Galβ4GlcNAc→)₂[GlcNAcβ→]Hex₃HexNAc₂dHex₁ 2028 Hex6HexNAc5 3 × Galβ4(Galβ4GlcNAc→)₃Hex₃HexNAc₂ CO 2067 Hex10HexNAc2 ManαGlc→(Manα→)₈Hex₁HexNAc₂ G Glc 2101 Hex5HexNAc4dHex3 GlcNAcβGlcNAcβ→Hex₅HexNAc₃dHex₃ CO, FC 2142 Hex4HexNAc5dHex3 Galβ4Galβ4GlcNAc→Hex₃HexNAc₄dHex₃ CO, FC, N > H 2174 Hex6HexNAc5dHex GlcNAcβGlcNAcβ→Hex₆HexNAc₄dHex₁ CO, F 3 × Galβ4 (Galβ4GlcNAc→)₃Hex₃HexNAc₂dHex₁2229 Hex11HexNAc2 Glc Glc₂→(Manα→)₈Hex₁HexNAc₂ G Manα 2304Hex5HexNAc5dHex3 GlcNAcβ GlcNAcβ→Hex₅HexNAc₄dHex₃ CO, FC, N═H 2320Hex6HexNAc5dHex2 GlcNAcβ GlcNAcβ→Hex₆HexNAc₄dHex₂ CO, FC 2393Hex7HexNAc6 Galβ4 Galβ4GlcNAc→Hex₆HexNAc₅ CO 2466 Hex6HexNAc5dHex3GlcNAcβ GlcNAcβ→Hex₆HexNAc₄dHex₃ CO, FC 2539 Hex7HexNAc6dHex GlcNAcβGlcNAcβ→Hex₇HexNAc₅dHex₁ CO, F 4 × Galβ4 (Galβ4GlcNAc→)₄Hex₃HexNAc₂dHex₁*[M + Na]⁺ ion, first isotope. ^(§)“→” indicates linkage to amonosaccharide in the rest of the structure; “[ ]” indicates branch inthe structure. ^(#)Preferred structure group based on monosaccharidecompositions according to the present invention. HI, high-mannose; LO,low-mannose; S, soluble mannosylated; HF, fucosylated high-mannose; G,glucosylated high-mannose; HY, hybrid-type or monoantennary; CO,complex-type; F, fucosylation; FC, complex fucosylation; N═H, terminalHexNAc (HexNAc = Hex); N > H, terminal HexNAc (HexNAc > Hex).

TABLE 31 Proposed composition m/z α-Man β-GlcNAc β4-Gal β3-GalHex2HexNAc 568 −−− −−− HexHexNAc2 609 +++ −−− Hex2HexNAcdHex 714 +++Hex3HexNAc 730 − HexHexNAc2dHex 755 +++ Hex2HexNAc2 771 ++ ++ − −Hex4HexNAc 892 −−− −−− Hex2HexNAc2dHex 917 − ++ − − Hex3HexNAc2 933 ++++ − − HexHexNAc3dHex 958 Hex2HexNAc3 974 +++ ++ −−− Hex5HexNAc 1054 −−−Hex3HexNAc2dHex 1079 −− ++ − − Hex4HexNAc2 1095 −− + − − Hex2HexNAc3dHex1120 +++ + −−− Hex3HexNAc3 1136 ++ −−− ++ −− Hex2HexNAc2dHex3 1209 −−−−−− Hex6HexNAc 1216 −−− +++ +++ Hex4HexNAc2dHex 1241 −−− − Hex5HexNAc21257 −− Hex3HexNAc3dHex 1282 ++ −−− + − Hex4HexNAc3 1298 +++ + − −Hex3HexNAc4 1339 +++ −−− Hex7HexNAc 1378 +++ +++ Hex5HexNAc2dHex 1403−−− − Hex6HexNAc2 1419 −− + Hex3HexNAc3dHex2 1428 +++ Hex4HexNAc3dHex1444 ++ − − Hex5HexNAc3 1460 + −− + − Hex3HexNAc4dHex 1485 −−− ++ −Hex4HexNAc4 1501 + −−− −− − Hex8HexNAc 1540 −−− − Hex3HexNAc5 1542 +++Hex6HexNAc2dHex 1565 −−− −−− Hex7HexNAc2 1581 −− Hex4HexNAc3dHex2 1590Hex5HexNAc3dHex 1606 −− −− − Hex6HexNAc3 1622 −− −− −− − Hex4HexNAc4dHex1647 −−− − Hex5HexNAc4 1663 − −− Hex3HexNAc5dHex 1688 −−− ++ −−−Hex4HexNAc5 1704 +++ Hex8HexNAc2 1743 −− Hex5HexNAc3dHex2 1752 −−− −−−Hex6HexNAc3dHex 1768 −− −− −− Hex7HexNAc3 1784 − −−− Hex4HexNAc4dHex21793 −−− ++ −−− Hex5HexNAc4dHex 1809 −− −−− Hex6HexNAc4 1825 +++ +++ −−Hex4HexNAc5dHex 1850 +++ Hex5HexNAc5 1866 −−− −−− Hex3HexNAc6dHex 1891++ −−− Hex9HexNAc2 1905 −−− Hex5HexNAc4dHex2 1955 −−− −− −Hex6HexNAc4dHex 1971 −−− −−− Hex7HexNAc4 1987 −−− −−− Hex4HexNAc5dHex21996 −−− +++ Hex5HexNAc5dHex 2012 −−− −− Hex6HexNAc5 2028 − −−− −Hex10HexNAc2 2067 −−− − Hex5HexNAc4dHex3 2101 − Hex6HexNAc4dHex2 2117−−− −−− Hex7HexNAc4dHex 2133 −−− −−− Hex4HexNAc5dHex3 2142 −−− −−−Hex6HexNAc5dHex 2174 −− −−− − Hex5HexNAc7 2272 +++ Hex5HexNAc5dHex3 2304−−− +++ Hex6HexNAc5dHex2 2320 −−− −−− Hex7HexNAc6 2393 −− −−−Hex6HexNAc5dHex3 2466 −−− −−− Hex7HexNAc6dHex 2539 −−− −−− Hex8HexNAc72758 −−− −−−

TABLE 32 Proposed composition m/z β4-Gal β-GlcNAc Hex2HexNAc 568 − −−−HexHexNAc2 609 +++ Hex3HexNAc 730 Hex2HexNAc2 771 −− Hex4HexNAc 892 −−−Hex2HexNAc2dHex 917 − Hex3HexNAc2 933 − Hex2HexNAc3 974 +++ Hex5HexNAc1054 Hex3HexNAc2dHex 1079 Hex4HexNAc2 1095 Hex2HexNAc3dHex 1120 +++Hex3HexNAc3 1136 ++ −−− Hex2HexNAc2dHex3 1209 −−− −−− Hex6HexNAc 1216Hex4HexNAc2dHex 1241 Hex5HexNAc2 1257 Hex3HexNAc3dHex 1282 + −−Hex4HexNAc3 1298 Hex3HexNAc4 1339 +++ Hex2HexNac2dHex4 1355 +++Hex7HexNAc 1378 Hex5HexNAc2dHex 1403 Hex6HexNAc2 1419 Hex4HexNAc3dHex1444 + Hex5HexNAc3 1460 ++ − Hex3HexNAc4dHex 1485 ++ −−− Hex4HexNAc41501 −− −−− Hex8HexNAc 1540 Hex3HexNAc5 1542 +++ Hex6HexNAc2dHex 1565Hex7HexNAc2 1581 Hex4HexNAc3dHex2 1590 +++ +++ Hex5HexNAc3dHex 1606 −Hex6HexNAc3 1622 −− − Hex4HexNAc4dHex 1647 −−− Hex5HexNAc4 1663 −−− ++Hex3HexNAc5dHex 1688 ++ −−− Hex9HexNAc 1702 −−− −−− Hex4HexNAc5 1704 +++−−− Hex8HexNAc2 1743 Hex5HexNAc3dHex2 1752 +++ Hex6HexNAc3dHex 1768 −Hex7HexNAc3 1784 −−− −−− Hex4HexNAc4dHex2 1793 +++ Hex5HexNAc4dHex 1809−−− + Hex4HexNAc5dHex 1850 −−− Hex3HexNAc6dHex 1891 ++ −−− Hex9HexNAc21905 Hex5HexNAc4dHex2 1955 −−− Hex4HexNAc5dHex2 1996 −−− Hex5HexNAc5dHex2012 −−− −−− Hex6HexNAc5 2028 −−− Hex10HexNAc2 2067 Hex5HexNAc4dHex32101 + Hex6HexNAc5dHex 2174 −−− Hex7HexNAc6 2393 −−− −−− Hex7HexNAc6dHex2539 −−− −−−

TABLE 33 Proposed composition m/z α-Man β4-Gal β-GlcNAc Hex2HexNAc 568−−− − −−− HexHexNAc2 609 +++ − −−− Hex3HexNAc 730 −− − HexHexNAc2dHex755 +++ −−− Hex2HexNAc2 771 ++ − −− Hex4HexNAc 892 −−− − −−−Hex2HexNAc2dHex 917 −− − −− Hex3HexNAc2 933 − − −− Hex2HexNAc3 974 ++ +−−− Hex5HexNAc 1054 −−− Hex3HexNAc2dHex 1079 −−− − −− Hex4HexNAc2 1095−− − − Hex2HexNAc3dHex 1120 ++ + −−− Hex3HexNAc3 1136 + ++ −− Hex6HexNAc1216 −− Hex4HexNAc2dHex 1241 −−− Hex5HexNAc2 1257 −−− Hex3HexNAc3dHex1282 + −− Hex4HexNAc3 1298 + Hex3HexNAc4 1339 ++ −−− Hex7HexNAc 1378 −−−Hex5HexNAc2dHex 1403 −−− Hex6HexNAc2 1419 −− Hex3HexNAc3dHex2 1428 +++Hex4HexNAc3dHex 1444 Hex5HexNAc3 1460 + Hex3HexNAc4dHex 1485 ++ −−−Hex4HexNAc4 1501 −− −−− Hex8HexNAc 1540 −−− −−− Hex3HexNAc5 1542 + ++−−− Hex6HexNAc2dHex 1565 −−− − Hex7HexNAc2 1581 −− Hex4HexNAc3dHex2 1590−−− ++ Hex5HexNAc3dHex 1606 − −− + Hex6HexNAc3 1622 −− −− ++Hex4HexNAc4dHex 1647 −− −−− Hex5HexNAc4 1663 −−− + Hex3HexNAc5dHex 1688++ −−− Hex4HexNAc5 1704 +++ Hex8HexNAc2 1743 −− Hex5HexNAc3dHex2 1752+++ Hex6HexNAc3dHex 1768 − −− + Hex7HexNAc3 1784 −−− −− Hex4HexNAc4dHex21793 + −−− Hex5HexNAc4dHex 1809 −−− Hex6HexNAc4 1825 −−− − +Hex4HexNAc5dHex 1850 −−− −−− Hex5HexNAc5 1866 −−− −−− Hex3HexNAc6dHex1891 −−− ++ −−− Hex9HexNAc2 1905 −−− Hex5HexNAc4dHex2 1955 ++Hex6HexNAc4dHex 1971 −−− + Hex7HexNAc4 1987 +++ Hex4HexNAc5dHex2 1996−−− Hex5HexNAc5dHex 2012 −−− −−− Hex6HexNAc5 2028 −−− Hex10HexNAc2 2067−−− Hex5HexNAc4dHex3 2101 + Hex6HexNAc5dHex 2174 −−− Hex6HexNAc6 2231−−− −−− Hex5HexNAc5dHex3 2304 −−− Hex6HexNAc5dHex2 2320 −−− −−−Hex6HexNAc6dHex 2377 −−− −−− Hex7HexNAc6 2393 −−− −− Hex6HexNAc5dHex32466 Hex7HexNAc6dHex 2539 −−− −−− Hex8HexNAc6dHex4 3140 −−− −−−

TABLE 34 Sialidase resistant acidic N-glycans in cord blood CD133+ andCD133− cells. m/z [M − H]⁻ CD133+/composition Hex3HexNAc2SP 989.28Hex4HexNAc3SP 1354.41 Hex4HexNAc3dHexSP 1500.47 Hex5HexNAc3SP 1516.46NeuAc2Hex2HexNAc3SP 1612.49 Hex4HexNAc4dHex4SP 1703.55 Hex5HexNAc4SP1719.54NeuAcNeuGcHex2HexNAc4SP/NeuAcNeuGcHex5HexNAc2/Hex5HexNAc2dHex3SP21831.57 Hex5HexNAc4dHexSP 1865.60 NeuAc2Hex3HexNAc4SP 1977.63Hex5HexNAc4dHex2SP 2011.66 Hex5HexNAc5dHexSP 2068.68 Hex6HexNAc5SP2084.67 Hex10HexNAc2SP/Hex7HexNAc4SP2/NeuAc2Hex3HexNAc4dHexSP 2123.64NeuAc2Hex6HexNAc3/NeuGc2Hex4HexNAc3dHex2/NeuAcHex3HexNAc5SP 2180.75Hex6HexNAc5dHexSP 2230.73 Hex7HexNAc5dSP 2246.73 NeuAc2Hex4HexNAc5SP2342.76 Hex6HexNAc5dHex2SP 2376.79 Hex6HexNAc6dHexSP 2433.81Hex7HexNAc6SP 2449.81 Hex7HexNAc6dHexSP 2595.86 Hex8HexNAc7dHexSP2960.99 CD133−/composition Hex4HexNAc2SP 1151.33 Hex3HexNAc3SP 1192.36Hex5HexNAc2SP 1313.38 Hex3HexNAc3dHexSP 1338.41 Hex4HexNAc3SP 1354.41Hex6HexNAc2SP/NeuAc2Hex2HexNac2dHex 1475.44 Hex4HexNAc3dHexSP 1500.47Hex5HexNAc3SP 1516.46 Hex3HexNAc4dHexSP 1541.49 Hex4HexNAc4SP 1557.49Hex4HexNAc4SP2/Hex7HexNAc2SP/NeuAc2Hex3HexNAc2dHex 1637.49Hex5HexNAc3dHexSP 1662.52 Hex6HexNAc3SP/NeuAc2Hex2HexNAc3dHex 1678.51Hex4HexNAc4dHexSP 1703.55 Hex5HexNAc4SP 1719.54 NeuAcHex4HexNAc3dHexSP1791.56 Hex5HexNAc4dHexSP 1865.60 Hex6HexNAc4SP 1881.65NeuAcHex5HexNAc4SP 2010.64 Hex5HexNAc4dHex2SP 2011.66 Hex5HexNAc5dHexSP2068.68 Hex6HexNAc5SP 2084.67NeuAcHex5HexNAc4dHexSP/NeuAcHex8HexNAc2dHex 2156.69 Hex5HexNAc4dHex3SP2157.71 Hex6HexNAc5dHexSP 2230.73 Hex6HexNAc5dHex2SP 2376.79Hex6HexNAc6dHexSP 2433.81 NeuAcHex6HexNAc5dHexSP/NeuAcHex9HexNAc3dHex2521.83 Hex6HexNAc5dHex3SP 2522.85 Hex7HexNAc6dHexSP 2595.86Hex8HexNAc7dHexSP 2960.99

TABLE 35 Reagent Target FES 22 FES 30 mEF FITC-PSA α-Man − − + FITC-RCAβ-Gal (Galβ4GlcNAc) + − +/− FITC-PNA β-Gal (Galβ3GalNAc) + + − FITC-MAAα2,3-sialyl-LN + + − FITC-SNA α2,6-sialyl-LN + n.d. + FITC-PWAI-antigen + + n.d. FITC-STA i-antigen + − + FITC-WFA β-GalNAc + + −NeuGc-PAA-biotin NeuGc-lectin + + + anti-GM3 (Gc) mAbNeuGcα3Galβ4Glc + + + FITC-LTA α-Fuc + − + FITC-UEA α-Fuc + − + mAb LexLewis^(x) + n.d. − mAb sLex sialyl-Lewis^(x) + n.d. − +, specificbinding. −, no specific binding. n.d., not determined.

TABLE 36 % of Lectins Target positive cells FITC-GNA α-Man 27.8 FITC-HHAα-Man 95.3 FITC-PSA α-Man 95.5 FITC-RCA β-Gal (Galβ4GlcNAc) 94.8FITC-PNA β-Gal (Galβ3GalNAc) 31.1 FITC-MAA α2,3-sialylation 89.9FITC-SNA α2,6-sialylation 14.3 FITC-PWA I-antigen 1.9 FITC-STA i-antigen11.9 FITC-LTA α-Fuc 2.8 FITC-UEA α-Fuc 8.0

TABLE 37 BM MSC lectin concentration, μg/ml Lectin Target 0.25 0.5 1 2.55 10 20 40 FITC-GNA α-Man −¹⁾ − ++ ++ ++ ++ ++ ++ FITC-HHA α-Man ++ +++++ +++ +++ +++ +++ +++ FITC-PSA α-Man ++ ++ ++ +++ +++ +++ +++ +++FITC-RCA β-Gal (Galβ4GlcNAc) − − +/− +/− + + ++ ++ FITC-PNA β-Gal(Galβ3GalNAc) − − − − +/− +/− +/− + FITC-MAA α2,3-sialylation − − −+/− + ++ ++ ++ FITC-SNA α2,6-sialylation − − − − +/− +/− + + FITC-PWAI-antigen − − − − − − +/− +/− FITC-STA i-antigen − − − − − +/− +/− +/−FITC-LTA α-Fuc − − − − − − − − FITC-UEA α-Fuc − − − +/− +/− + ++ ++FITC-MBL α-Man/β-GlcNAc − − − − − − +/− + ¹⁾Grading ofstaining/labelling: +++ very intense, ++ intense, + low, +/− barelydetectable, − not labelled.

TABLE 38 The 15 characteristic neutral N-glycan signals of the hESCN-glycome. The signals are expressed in all the analyzed hESC samplesand they are listed in order of relative abundance. The proposedstructural classification is as described in the Examples. m/z ProposedProposed No [M + Na]⁺ composition classification 1. 1905.6 H₉N₂high-mannose 2. 1419.5 H₆N₂ high-mannose 3. 1743.6 H₈N₂ high-mannose 4.1257.4 H₅N₂ high-mannose 5. 1581.5 H₇N₂ high-mannose 6. 1079.4 H₃N₂F₁low-mannose 7. 2067.7 H₁₀N₂ other types 8. 1095.4 H₄N₂ low-mannose 9.933.3 H₃N₂ low-mannose 10. 1663.6 H₅N₄ complex-type 11. 1622.6 H₆N₃hybrid/monoantennary 12. 1809.6 H₅N₄F₁ complex-type 13. 1460.5 H₅N₃hybrid/monoantennary 14. 1485.5 H₃N₄F₁ complex-type; terminal N (N > H)15. 1444.5 H₄N₃F₁ hybrid/monoantennary

TABLE 39 The 15 characteristic acidic N-glycan signals of the hESCN-glycome. The signals are expressed in all the analyzed hESC samplesand they are listed in order of relative abundance. The proposedstructural classification is as described in the Examples. m/z ProposedProposed No [M − H]⁻ composition classification 1. 2076.7 S₁H₅N₄F₁complex-type 2. 2222.8 S₁H₅N₄F₂ complex-type; complex fucosylation 3.2367.8 S₂H₅N₄F₁ complex-type 4. 1930.7 S₁H₅N₄ complex-type 5. 2441.9S₁H₆N₅F₁ complex-type 6. 2092.7 G₁H₅N₄F₁ complex-type 7. 2117.8 S₁H₄N₅F₁complex-type; terminal N (N > H) 8. 2587.9 S₁H₆N₅F₂ complex-type;complex fucosylation 9. 2368.9 S₁H₅N₄F₃ complex-type; complexfucosylation 10. 2263.8 S₁H₄N₅F₂ complex-type; complex fucosylation;terminal N (N > H) 11. 1711.6 S₁H₄N₃F₁ hybrid/monoantennary 12. 2279.8S₁H₅N₅F₁ complex-type; terminal N (N = H) 13. 2238.8 G₁H₅N₄F₂complex-type; complex fucosylation 14. 2733.0 S₂H₆N₅F₁ complex-type 15.2807.0 S₁H₇N₆F₁ complex-type

TABLE 40 Neutral and acidic N-glycan signals expressed exclusively inthe four hESC samples. The signals are listed in order of increasing m/z(molecular mass) of the detected signals, first neutral N-glycans andthen acidic N-glycans. The proposed structural classification is asdescribed in the Examples. Neutral m/z Proposed Proposed (5) [M + Na]⁺composition classification 1501.5 H₄N₄ complex-type 1590.6 H₄N₃F₂hybrid/monoantennary; complex fucosylation 1793.6 H₄N₄F₂ complex-type;complex fucosylation 1825.6 H₆N₄ complex-type 2320.8 H₆N₅F₂complex-type; complex fucosylation Acidic m/z Proposed Proposed (14) [M− H]⁻ composition classification 1500.5 H₄N₃F₁P₁ hybrid/monoantennary;fucosylated 2174.8 S₁H₄N₆ complex-type; terminal N (N > H) 2263.8S₁H₄N₅F₂ complex-type; terminal N (N > H); complex fucosylation 2457.9S₁H₇N₅ complex-type 2660.9 S₁H₇N₆ complex-type 2953.1 S₁H₇N₆F₂complex-type; complex fucosylation 3172.1 S₁H₈N₇F₁ complex-type 3245.2S₁H₇N₆F₄ complex-type; complex fucosylation 3317.2 S₂H₈N₇ complex-type3463.2 S₂H₈N₇F₁ complex-type 3608.3 S₃H₈N₇ complex-type 3610.3 S₁H₈N₇F₄complex-type; complex fucosylation 3682.3 S₂H₉N₈ complex-type 3756.3S₁H₁₀N₉ complex-type

TABLE 41 N-glycan structural feature analysis based on proposedmonosaccharide compositions of four hESC lines FES 21, FES 22, FES 29,and FES 30. FES 21* FES 22 FES 29 FES 30 EB st.3 Neutral A N = 2 and 5 ≦H ≦ 10 high-mannose type 84^(#) 73 79 79 73 72 N-glycans B N = 2 and 1 ≦H ≦ 4 low-mannose type 5 11 7 8 12 12 C N = 3 and H ≧ 2hybrid/monoantennary 3 7 3 3 5 6 D N ≧ 4 and H ≧ 3 complex-type 6 9 1010 8 8 E other types 2 0 1 0 2 2 N ≧ 3 F F ≧ 1 fucosylation 8 11 10 1014 15 G F ≧ 2 complex fucosylation 1 0 2 2 2 2 H^(§) N > H ≧ 2 terminalN (N > H) 1 2 1 1 3 3 I N = H ≧ 5 terminal N (N = H) 0 2 0 0 1 1Sialylated J N = 3 and H ≧ 3 hybrid/monoantennary 8 2 5 9 13 14N-glycans K N ≧ 4 and H ≧ 3 complex-type 91  98 94 90 83 77 L othertypes 1 0 1 1 4 9 N ≧ 3 M F ≧ 1 fucosylation 85  96 75 78 83 86 N F ≧ 2complex fucosylation 24  34 23 19 12 11 O N > H ≧ 3 terminal N (N > H)10  8 6 5 10 10 P N = H ≧ 5 terminal N (N = H) 3 4 4 2 14 20 The numbersrefer to percentage from either neutral (A-E) or acidic (J-L) N-glycanpools, or from subfractions of hybrid/monoantenary and complex-typeN-glycans (N ≧ 3, F-I and M-P). EB 29 and EB 30: embryoid bodies derivedfrom hESC lines FES 29 and FES 30, respectively; st.3 29: stage 3differentiated cells derived from hESC line FES 29. H: hexose; N:N-acetylhexosamine; F: deoxyhexose.

TABLE 42¹⁾ FES 21 FES 22 FES 29 FES 30 EB²⁾ Affymetrix ID Gene Bank IDGene Det.³⁾ Ch.⁴⁾ Det. Ch. Det. Ch. Det. Ch. Det. 206109_at NM_000148.1FUT1 P I P I P I P I A 214088_s_at AW080549 FUT3 M NC A NC A NC A NC A209892_at AF305083.1 FUT4 P I P I P I P I A 211225_at U27330 FUT5 A NC ANC A NC A NC A 211225_at U27329.1 FUT5 A NC A NC A NC A NC A 210399_x_atU27336.1 FUT6 A NC A NC A NC A NC A 211882_x_at U27331.1 FUT6(1) A NC ANC A NC A NC A 211885_x_at U27332.1 FUT6(2) A NC A NC A NC A NC A211465_x_at U27335.1 FUT6(minor) A NC A NC A NC A NC A 210506_atU11282.1 FUT7 A NC A NC A NC A NC A 203988_s_at NM_004480.1 FUT8 P NC PNC P NC P NC A 207696_at NM_006581.1 FUT9 A NC A NC A NC A NC A229203_at NM_173593 β4GalNAc-T3 A NC A NC A NC A NC A 200016_x_atNM_002409 MGAT3 P NC P D P D P D P 208058_s_at NM_002409.2 MGAT3 A NC ANC A NC A NC A 209764_at AL022312 β4GlcNAcT A NC A MD A MD A NC A206435_at NM_001478.2 GALGT A NC A NC A NC A NC A 206720_at NM_002410.2MGAT5 A NC A NC A NC A NC A 203102_s_at NM_002408.2 MGAT2 P I P NC P I PI P 201126_s_at NM_002406.2 MGAT1 P NC P NC P NC P NC P 219797_atNM_012214.1 GNT4a A NC P NC A NC M NC A 220189_s_at NM_014275.1 GNT4b PD P NC P NC P NC P 204856_at AB049585 β3GlcNAc-T3 A NC A NC A NC A NC A225612_s_at BE672260 β3GlcNAc-T5 P D P D P D P D P 232337_at XM_091928β3GlcNAc-T7 P NC P NC P NC P NC A 221240_s_at NM_030765.1 β3GlcNAc-T4 PNC A NC A NC P NC A 204856_at NM_014256.1 β3GnT3 A NC A NC A NC A NC A205505_at NM_001490.1 β6GlcNAcT P I P NC P NC A NC A 203188_atNM_006876.1 i β3GlcNAcT P D P D P MD P NC P 211020_at L19659.1 Iβ6GlcNAcT A NC M NC A NC A NC A 214504_at NM_020459.1 A α3GalNAcT A NC ANC A NC A NC A 211812_s_at AB050856.1 globosideT P NC A NC P NC P NC A221131_at NM_016161.1 α4GlcNAcT M NC P NC P NC M NC A 221935_s_at AER61P I P I P I P I A 225689_at AGO61 P NC P NC P NC P NC P 210571_s_at CMAHA NC A NC A NC A NC A 205518_s_at CMAH A D M NC A D A NC P 213355_atST3GAL6 A NC A NC A NC A NC A 211379_x_at β3GALT3 P D P D P NC P D P218918_at MAN1C1 P NC P NC P NC P NC P 208450_at LGALS2 A NC A NC A NC ANC A 208949_s_at LGALS3 P D P D P D P D P ¹⁾Data reference: Skottman,H., et al. (2005). ²⁾EB, embryoid bodies used as reference incalculation of fold changes. ³⁾Det. (detection) codes: P, present; A,absent; M, medium. ⁴⁾Ch. (fold change) codes: I, increased; D,decreased; NC, no change.

TABLE 43 % m/z proposed composition % m/z proposed composition hEFneutral N-glycans mEF neutral N-glycans 19.5¹⁾ 1743 Hex8HexNAc2 13.71905 Hex9HexNAc2 17.1 1905 Hex9HexNAc2 13.5 1419 Hex6HexNAc2 16.2 1419Hex6HexNAc2 13.5 1743 Hex8HexNAc2 12.6 1581 Hex7HexNAc2 11.2 1581Hex7HexNAc2 4.6 1257 Hex5HexNAc2 10.3 1257 Hex5HexNAc2 3.6 1079Hex3HexNAc2dHex1 2.7 1054 Hex5HexNAc1 2.1 2067 Hex10HexNAc2 2.6 568Hex2HexNAc1 2.4 2067 Hex10HexNAc2 2.1 1216 Hex6HexNAc1 2.0 892Hex4HexNAc1 1.9 933 Hex3HexNAc2 hEF acidic N-glycans mEF acidicN-glycans 23.0²⁾ 2076 NeuAc1Hex5HexNAc4dHex1 30.3 2238NeuAc1Hex6HexNAc4dHex1 8.5 2367 NeuAc2Hex5HexNAc4dHex1 12.4 2076NeuAc1Hex5HexNAc4dHex1 8.1 2441 NeuAc1Hex6HexNAc5dHex1 9.8 2092NeuAc1Hex6HexNAc4 6.0 2221 NeuAc2Hex5HexNAc4 5.9 1930 NeuAc1Hex5HexNAc45.9 1930 NeuAc1Hex5HexNAc4 2.6 2367 NeuAc2Hex5HexNAc4dHex1 5.3 2733NeuAc1Hex6HexNAc5dHex3 2.6 1914 NeuAc1Hex4HexNAc4dHex1 3.5 2368NeuAc1Hex5HexNAc4dHex3 2.0 1727 NeuAc1Hex5HexNAc3 2.9 2732NeuAc2Hex6HexNAc5dHex1 1.9 1889 NeuAc1Hex6HexNAc3 2.5 3391NeuAc1Hex9HexNAc8 1.7 2221 NeuAc2Hex5HexNAc4 2.5 3098NeuAc2Hex7HexNAc6dHex1 1.6 2441 NeuAc1Hex6HexNAc5dHex1 ¹⁾Together thetabled signals comprise over 75% of total signal intensity. ²⁾Togetherthe tabled signals comprise over 67% of total signal intensity.

TABLE 44 Neutral N-glycan structures proportion, of feeder cells %proposed composition proposed structure types hEF mEF Hex₅₋₁₃HexNAc₂high-mannose/glucosylated 76 72 Hex₁₋₄HexNAc₂dHex₀₋₁ low-mannose 8 7n_(HexNAc) = 3 ja n_(Hex) ≧ 2 hybrid/monoantennary 4 6 n_(HexNAc) ≧ 4 jan_(Hex) ≧ 2 complex-type 9 11 other types 3 4 n_(dHex) ≧ 1 fucosylation13 8 n_(dHex) ≧ 2 complex fucosylation 0.5 0.2 n_(HexNAc) > n_(Hex) ≧ 2terminal HexNAc, N > H¹⁾ 2 2 n_(HexNAc) = n_(Hex) ≧ 5 terminal HexNAc, N= H — 0.3 ¹⁾N, HexNAc; H, Hex.

TABLE 45 Acidic N-glycan structures proportion, of feeder cells %proposed composition proposed structure types hEF mEF n_(HexNAc) = 3 jan_(Hex) ≧ 5 hybrid-type 3 8 n_(HexNAc) = 3 ja n_(Hex) = 3-4monoantennary 4 6 n_(HexNAc) ≧ 4 ja n_(Hex) ≧ 3 complex-type 92 86 muut— 1 0 n_(dHex) ≧ 1 fucosylation 76 67 n_(dHex) ≧ 2 complex fucosylation21 4 n_(HexNAc) > n_(Hex) ≧ 2 terminal HexNAc, N > H¹⁾ 1 2 n_(HexNAc) =n_(Hex) ≧ 5 terminal HexNAc, N = H 1.5 1.5 NeuAc + 16 Da NeuGc — — +80Da sulphate/phosphate ester 1 9 ¹⁾N, HexNAc; H, Hex.

TABLE 46 Proposed composition m/z hESC EB st.3 hEF mEF BM MSC OB CB MSCAC CB MNC CD 34+ CD 133+ LIN− CD 8− Hex₅₋₉HexNAc₂ (includinghigh-mannose type N-glycans) Hex5HexNAc21257 + + + + + + + + + + + + + + Hex6HexNAc21419 + + + + + + + + + + + + + + Hex7HexNAc21581 + + + + + + + + + + + + + + Hex8HexNAc21743 + + + + + + + + + + + + + + Hex9HexNAc21905 + + + + + + + + + + + + + + Hex₁₋₄HexNAc₂dHex₀₋₁ (includinglow-mannose type N-glycans) HexHexNAc2 609 + + + + + + + +HexHexNAc2dHex 755 + + + + + Hex2HexNAc2 771 + + + + + + + + + + + + + +Hex2HexNAc2dHex 917 + + + + + + + + + + + + + + Hex3HexNAc2933 + + + + + + + + + + + + + + Hex3HexNAc2dHex1079 + + + + + + + + + + + + + + Hex4HexNAc21095 + + + + + + + + + + + + + + Hex4HexNAc2dHex1241 + + + + + + + + + + + + + + Hex₁₀₋₁₂HexNAc₂ (including glucosylatedhigh- mannose type N-glycans) Hex10HexNAc22067 + + + + + + + + + + + + + + Hex11HexNAc2 2229 + + + + + + + + + + +Hex12HexNAc2 2391 + + + + + + + + + + Hex₅₋₉HexNAc₂dHex₁ (includingfucosylated high- mannose type N-glycans) Hex5HexNAc2dHex1403 + + + + + + + + + + + + + + Hex6HexNAc2dHex1565 + + + + + + + + + + Hex7HexNAc2dHex 1727 + Hex₁₋₉HexNAc₁ (includingsoluble glycans) Hex2HexNAc 568 + + + + + + + Hex3HexNAc730 + + + + + + + + + Hex4HexNAc 892 + + + + + + + + + + + + + +Hex5HexNAc 1054 + + + + + + + + + + + + + + Hex6HexNAc1216 + + + + + + + + + + + + + + Hex7HexNAc1378 + + + + + + + + + + + + + + Hex8HexNAc1540 + + + + + + + + + + + + + Hex9HexNAc 1702 + + + + + + + + + +HexNAc = 3 and Hex ≧ 2 (including hybrid-type and monoantennaryN-glycans) Hex2HexNAc3 974 + + + Hex2HexNAc3dHex 1120 + + + + + + + + +Hex3HexNAc3 1136 + + + + + + + + + + + + + + Hex2HexNAc3dHex2 1266 +Hex3HexNAc3dHex 1282 + + + + + + + + + + + + + + Hex4HexNAc31298 + + + + + + + + + + + + + + Hex3HexNAc3dHex2 1428 + + + + + +Hex4HexNAc3dHex 1444 + + + + + + + + + + + + + + Hex5HexNAc31460 + + + + + + + + + + + + + + Hex4HexNAc3dHex2 1590 + + + + + + + + +Hex5HexNAc3dHex 1606 + + + + + + + + + + + + + + Hex6HexNAc31622 + + + + + + + + + + + + + + Hex5HexNAc3dHex2 1752 + + + +Hex6HexNAc3dHex 1768 + + + + + + + + + Hex7HexNAc3 1784 + + + + + + +Hex8HexNAc3 1946 + + HexNAc ≧ 4 and Hex ≧ 3 (including complex-type N-glycans) Hex3HexNAc4 1339 + + + + + + + + Hex3HexNAc4dHex1485 + + + + + + + + + + + + + + Hex4HexNAc4 1501 + + + + + + + + + +Hex3HexNAc5 1542 + + + + + + + + Hex4HexNAc4dHex1647 + + + + + + + + + + + + + + Hex5HexNAc41663 + + + + + + + + + + + + + + Hex3HexNAc5dHex1688 + + + + + + + + + + + + + + Hex4HexNAx51704 + + + + + + + + + + + + Hex4HexNAc4dHex2 1793 + + + + + + + +Hex5HexNAc4dHex 1809 + + + + + + + + + + + + + + Hex6HexNAc41825 + + + + + + + + + + + Hex4HexNAc5dHex 1850 + + + + + + +Hex5HexNAc5 1866 + + + + + + + + + + + + Hex3HexNAc6dHex 1891 + + + + +Hex5HexNAc4dHex2 1955 + + + + + + + + + + + Hex6HexNAc4dHex1971 + + + + + + + + Hex7HexNAc4 1987 + + + + + + + Hex4HexNAc5dHex21996 + + + + + + + Hex5HexNAc5dHex 2012 + + + + + + + + Hex6HexNAc52028 + + + + + + + + + + + Hex5HexNAc4dHex3 2101 + + + + + + + + + + +Hex6HexNAc4dHex2 2117 + + Hex7HexNAc4dHex 2133 + + + + Hex4HexNAc5dHex32142 + + + + + + + Hex8HexNAc4 2149 + + + + + Hex5HexNAc5dHex22158 + + + + Hex6HexNAc5dHex 2174 + + + + + + + + + + Hex7HexNAc52190 + + Hex6HexNAc6 2231 + + Hex7HexNAc4dHex2 2279 + + Hex5HexNAc5dHex32304 + + + Hex6HexNAc5dHex2 2320 + + + + + + Hex7HexNAc5dHex 2336 + +Hex8HexNAc5 2352 + + Hex7HexNAc6 2393 + + + + + + Hex7HexNAc4dHex32425 + + Hex6HexNAc5dHex3 2466 + + + Hex8HexNAc5dHex 2498 + +Hex7HexNAc6dHex 2539 + + + + + Hex6HexNAc5dHex4 2612 + + Hex8HexNAc72758 + + HexNAc ≧ 3 and dHex ≧ 1 (including fucosylated N- glycans)Hex2HexNAc3dHex 1120 + + + + + + + + + Hex2HexNAc3dHex2 1266 +Hex3HexNAc3dHex 1282 + + + + + + + + + + + + + + Hex3HexNAc3dHex21428 + + + + + + Hex4HexNAc3dHex 1444 + + + + + + + + + + + + + +Hex4HexNAc3dHex2 1590 + + + + + + + + + Hex5HexNAc3dHex1606 + + + + + + + + + + + + + + Hex5HexNAc3dHex2 1752 + + + +Hex6HexNAc3dHex 1768 + + + + + + + + + Hex3HexNAc4dHex1485 + + + + + + + + + + + + + + Hex4HexNAc4dHex1647 + + + + + + + + + + + + + + Hex3HexNAc5dHex1688 + + + + + + + + + + + + + + Hex4HexNAc4dHex2 1793 + + + + + + + +Hex5HexNAc4dHex 1809 + + + + + + + + + + + + + + Hex4HexNAc5dHex1850 + + + + + + + Hex3HexNAc6dHex 1891 + + + + + Hex5HexNAc4dHex21955 + + + + + + + + + + + Hex6HexNAc4dHex 1971 + + + + + + + +Hex4HexNAc5dHex2 1996 + + + + + + + Hex5HexNAc5dHex 2012 + + + + + + + +Hex5HexNAc4dHex3 2101 + + + + + + + + + + + Hex6HexNAc4dHex2 2117 + +Hex7HexNAc4dHex 2133 + + + + Hex4HexNAc5dHex3 2142 + + + + + + +Hex5HexNAc5dHex2 2158 + + + + Hex6HexNAc5dHex 2174 + + + + + + + + + +Hex7HexNAc4dHex2 2279 + + Hex5HexNAc5dHex3 2304 + + + Hex6HexNAc5dHex22320 + + + + + + Hex7HexNAc5dHex 2336 + + Hex7HexNAc4dHex3 2425 + +Hex6HexNAc5dHex3 2466 + + + Hex8HexNAc5dHex 2498 + + Hex7HexNAc6dHex2539 + + + + + Hex6HexNAc5dHex4 2612 + + HexNAc ≧ 3 and dHex ≧ 2(including multifucosylated N-glycans) Hex2HexNAc3dHex2 1266 +Hex3HexNAc3dHex2 1428 + + + + + + Hex4HexNAc3dHex21590 + + + + + + + + + Hex5HexNAc3dHex2 1752 + + + + Hex4HexNAc4dHex21793 + + + + + + + + Hex5HexNAc4dHex2 1955 + + + + + + + + + + +Hex4HexNAc5dHex2 1996 + + + + + + + Hex5HexNAc4dHex32101 + + + + + + + + + + + Hex6HexNAc4dHex2 2117 + + Hex4HexNAc5dHex32142 + + + + + + + Hex5HexNAc5dHex2 2158 + + + + Hex7HexNAc4dHex22279 + + Hex5HexNAc5dHex3 2304 + + + Hex6HexNAc5dHex2 2320 + + + + + +Hex7HexNAc4dHex3 2425 + + Hex6HexNAc5dHex3 2466 + + + Hex6HexNAc5dHex42612 + + HexNAc > Hex ≧ 2 (terminal HexNAc, N > H) Hex2HexNAc3 974 + + +Hex2HexNAc3dHex 1120 + + + + + + + + + Hex2HexNAc3dHex2 1266 +Hex3HexNAc4 1339 + + + + + + + + Hex3HexNAc4dHex1485 + + + + + + + + + + + + + + Hex3HexNAc5 1542 + + + + + + + +Hex3HexNAc5dHex 1688 + + + + + + + + + + + + + + Hex4HexNAx51704 + + + + + + + + + + + + Hex4HexNAc5dHex 1850 + + + + + + +Hex3HexNAc6dHex 1891 + + + + + Hex4HexNAc5dHex2 1996 + + + + + + +Hex4HexNAc5dHex3 2142 + + + + + + + HexNAc = Hex ≧ 5 (terminal HexNAc, N= H) Hex5HexNAc5 1866 + + + + + + + + + + + + Hex5HexNAc5dHex2012 + + + + + + + + Hex5HexNAc5dHex2 2158 + + + + Hex6HexNAc6 2231 + +Hex5HexNAc5dHex3 2304 + + + hESC, human embryonic stem cells; EB,embryoid bodies derived from hESC; st.3, stage 3 differentiated cellsderived from hESC; hEF, human fibroblast feeder cells; mEF, murinefibroblast feeder cells; BM MSC, bone-marrow derived mesenchymal stemcells; OB, Osteoblast-differentiated cells derived from BM MSC; CB MSC,cord blood derived mesenchymal stem cells; OB, adipocyte-differentiatedcells derived from CB MSC; CB MNC, cord blood mononuclear cells; CD34+,CD133+, LIN−, and CD8−: subpopulations of CB MNC.

TABLE 47 BM CB CB Proposed composition m/z hESC EB st.3 hEF mEF MSC OBMSC AC MNC CD 34+ CD 133+ LIN− CD 8− HexNAc = 3 and Hex ≧ 2 (includinghybrid-type and monoantennary N-glycans) Hex3HexNAc3dHexSP 1338 +Hex4HexNAc3SP 1354 + + NeuAcHex3HexNAc3 1403 + + + + + + + + + +NeuGcHex3HexNAc3 1419 + Hex4HexNAc3dHexSP 1500 + + + + + + + + + +Hex5HexNAc3SP 1516 + + + + NeuAcHex3HexNAc3dHex1549 + + + + + + + + + + + + NeuAcHex3HexNAc3SP2 1563 + +NeuAcHex4HexNAc3 1565 + + + + + + + + + + + + + NeuGcHex4HexNAc31581 + + + + + Hex4HexNAc3dHex2SP 1646 + + Hex5HexNAc3dHexSP 1662 +Hex6HexNAc3SP and/or 1678 + + + + + + + + + + + + +NeuAc2Hex2HexNAc3dHex NeuAc2Hex3HexNAc3 1694 + NeuAcHex3HexNAc3dHexSP21709 + + NeuAcHex4HexNAc3dHex 1711 + + + + + + + + + + + + + +NeuAcHex5HexNAc3 and/or 1727 + + + + + + + + + + + + +NeuGcHex4HexNAc3dHex NeuGcHex5HexNAc3 1743 + NeuAcHex4HexNAc3dHexSP1791 + + + + + + Hex5HexNAc3dHex2SP 1808 + NeuAc2Hex3HexNAc3dHex1840 + + + + + + + NeuAc2Hex4HexNAc3 1856 + + NeuAcHex4HexNAc3dHex21857 + + NeuAcHex5HexNAc3dHex and/or 1873 + + + + + + + + + + + + + +NeuGcHex4HexNAc3dHex2 NeuAcHex6HexNAc3 1889 + + + + + + + + + + + + +Hex8HexNAc3SP and/or 2002 + + + + + + + + + + NeuAc2Hex4HexNAc3dHexNeuAcHex4HexNAc3dHex3 2003 + + NeuAc2Hex5HexNAc3 and/or2018 + + + + + + + NeuGcNeuAcHex4HexNAc3dHex NeuAcHex5HexNAc3dHex22019 + + + NeuGcNeuAcHex5HexNAc3 and/or 2034 + NeuGc2Hex4HexNAc3dHexNeuAcHex6HexNAc3dHex 2035 + + + + + + + + + + NeuGc2Hex5HexNAc3 2050 +NeuAcHex7HexNAc3 2051 + + + + + + NeuAc2Hex4HexNAc3dHexSP and/or2082 + + + Hex8HexNAc3SP2 NeuAcHex6HexNAc3dHexSP 2115 +Hex8HexNAc3dHexSP and/or 2148 + NeuAc2Hex4HexNAc3dHex2NeuAcHex8HexNAc3SP and/or 2293 + NeuAc3Hex4HexNAc3dHexNeuAc2Hex5HexNAc3dHex2 and/or 2310 + NeuGcNeuAcHex4HexNAc3dHex3NeuAc3Hex5HexNAc3SP 2389 + NeuAc2Hex5HexNAc3dHex2SP2390 + + + + + + + + + + NeuAc2Hex6HexNAc3dHexSP 2406 + + +NeuAcHex8HexNAc3dHexSP and/or 2439 + NeuAc3Hex4HexNAc3dHex2NeuAcHex9HexNAc3dHex 2521 + HexNAc ≧ 4 and Hex ≧ 3 (includingcomplex-type N- glycans) Hex4HexNAc4SP 1557 + + + + NeuAcHex3HexNAc41606 + Hex4HexNAc4SP2 1637 + + + + + + + + Hex4HexNAc4dHexSP 1703 + + +Hex4HexNAc4SP3 and/or 1717 + Hex7HexNAc2SP2 Hex5HexNAc4SP1719 + + + + + + NeuAcHex3HexNAc4dHex 1752 + NeuAcHex4HexNAc41768 + + + + + + + + + + + + NeuGcHex4HexNAc4 1784 + + Hex5HexNAc4SP2and/or 1799 + + + Hex8HexNAc2SP NeuAcHex3HexNAc5 1809 + NeuGcHex3HexNAc51825 + + Hex5HexNAc4dHexSP 1865 + + + + + + + + + + + Hex6HexNAc4SP1881 + Hex4HexNAc5dHexSP 1906 + + NeuAcHex4HexNAc4dHex1914 + + + + + + + + + + + + + NeuAcHex4HexNAc4SP2 1928 + +NeuAcHex5HexNAc4 1930 + + + + + + + + + + + + + + NeuGcHex5HexNAc41946 + + + + + + + + NeuAcHex4HexNAc5 1971 + + + + + + +NeuAcHex5HexNAc4Ac 1972 + Hex5HexNAc5SP2 2002 + + + + + + +NeuAcHex5HexNAc4SP 2010 + + Hex5HexNAc4dHex2SP 2011 + NeuGcHex5HexNAc4SP2026 + Hex6HexNAc4dHexSP 2027 + + Hex7HexNAc4SP and/or 2043 +Hex4HexNAc6SP2 and/or NeuAc2Hex3HexNAc4dHex NeuAcHex4HexNAc5SP2051 + + + + + Hex4HexNAc5dHex2SP 2052 + + + + NeuAc2Hex4HexNAc42059 + + NeuAcHex4HexNAc4dHex2 2060 + + + + + + NeuAcHex4HexNAc4dHexSP22074 + + NeuAcHex5HexNAc4dHex 2076 + + + + + + + + + + + + + +NeuAcHex6HexNAc4 and/or 2092 + + + + + + + + + + + +NeuGcHex5HexNAc4dHex NeuAcHex3HexNAc5dHex2 and/or 2101 +NeuAc2Hex4HexNAc4Ac NeuGcHex6HexNAc4 2108 + NeuAcHex4HexNAc5dHex2117 + + + + + + + + + Hex4HexNAc5dHex2SP2 2132 + NeuAcHex5HexNAc52133 + + + + + + + + + + NeuAc2Hex4HexNAc4SP 2139 NeuAcHex5HexNAc4dHexSP2156 + + + + + + + Hex5HexNAc4dHex3SP 2157 + Hex6HexNAc5SP2 2164 + + +Hex6HexNAc4dHex2SP and/or 2173 + Hex3HexNAc6dHex2SP2 NeuAcHex4HexNAc62174 + + + + + + NeuAc3Hex3HexNAc4 and/or 2188 + + NeuGcHex6HexNAc4SPand/or NeuAc2NeuGcHex2HexNAc4dHex NeuAc2Hex3HexNAc4dHex2 and/or 2189 + +Hex7HexNAc4dHexSP and/or Hex4HexNAc6dHexSP2 NeuAc2Hex4HexNAc4dHex 2205 +NeuAc2Hex4HexNAc4SP2 2219 + NeuAc2Hex5HexNAc42221 + + + + + + + + + + + + + + NeuAcHex5HexNAc4dHex22222 + + + + + + + + + + + + + + Hex6HexNAc5dHexSP 2230 + + + +NeuGcNeuAcHex5HexNAc4 2237 + + + + + + + NeuAcHex6HexNAc4dHex and/or2238 + + + + + + + + + + + + + + NeuGcHex5HexNAc4dHex2NeuAc2Hex3HexNAc5dHex and/or 2246 + + + + Hex7HexNAc5SPNeuGc2Hex5HexNAc4 2253 + + + + + + NeuAcHex7HexNAc4 and/or2254 + + + + + + + + + + NeuGcHex6HexNAc4dHex NeuAc2Hex4HexNAc5 2262 +NeuAcHex4HexNAc5dHex2 and/or 2263 + + + NeuAc2Hex5HexNAc4AcNeuAcHex5HexNAc5dHex 2279 + + + + + + + + + + + + + +NeuAc2Hex4HexNAc4dHexSP and/or 2285 + Hex11HexNAc2SP NeuAcHex6HexNAc52295 + + + + + + + + + + + + + NeuAc2Hex5HexNAc4SP 2301 +NeuAcHex5HexNAc4dHex2SP 2302 + NeuAc2Hex5HexNAc4Ac2 2305 +Hex6HexNAc4dHex3SP and/or 2319 + + + NeuGcNeuAcHex3HexNAc6NeuAcHex4HexNAc6dHex 2320 + + NeuAcHex5HexNAc5dHexAc 2321 + +Hex7HexNAc4dHex2SP and/or 2335 + + Hex4HexNAc6dHex2SP2 NeuAcHex5HexNAc62336 + + NeuAc3Hex4HexNac4 2350 + NeuAc2Hex4HexNAc4dHexSP 2365 + + +NeuAc2Hex5HexNAc4dHex 2367 + + + + + + + + + + + + + +NeuAcHex5HexNAc4dHex3 2368 + + + + + + + + + + + + + NeuAc2Hex6HexNAc4and/or 2383 + + + + + + + + + NeuGcNeuAcHex5HexNAc4dHexNeuAcHex6HexNAc4dHex2 and/or 2384 + + + + + + + NeuGcHex5HexNAc4dHex3NeuAc2Hex3HexNAc5dHex2 and/or 2392 + + Hex7HexNAc5dHexSPNeuAcHex3HexNAc5dHex4 2393 + NeuGc2Hex5HexNAc4dHex 2399 + + +NeuAcHex4HexNAc6dHexSP and/or 2400 + NeuGcHex6HexNAc4dHex2 and/orNeuAcHex7HexNAc4dHex NeuAc2Hex4HexNAc5dHex 2408 + + +NeuAcHex4HexNAc5dHex3 and/or 2409 + + NeuAc2Hex5HexNAc4dHexAcNeuAc2Hex5HexNAc5 2424 + + + + + NeuAcHex5HexNAc5dHex22425 + + + + + + + + + + NeuAcHex6HexNAc5dHex2441 + + + + + + + + + + + + + + NeuAc2Hex5HexNAc4dHexSP2447 + + + + + + + NeuAcHex5HexNAc4dHex3SP 2448 + + + + +NeuAcHex7HexNAc5 and/or 2457 + + + + + NeuGcHex6HexNAc5dHexNeuGcHex7HexNAc5 2473 + + NeuAcHex5HexNAc6dHex 2482 +NeuAcHex4HexNAc5dHex3SP 2489 + + Hex6HexNAc7SP 2490 + NeuAc3Hex5HexNAc42512 + + + + NeuAc2Hex5HexNAc4dHex2 2513 + + + + + + +NeuAcHex5HexNAc4dHex4 2514 + + NeuAcHex6HexNAc5dHexSP and/or2521 + + + + NeuAc3Hex2HexNAc5dHex2 Hex6HexNAc5dHex3SP 2522 + +NeuGcNeuAc2Hex5HexNAc4 2528 + + + + + NeuAc2Hex6HexNAc4dHex and/or2529 + + + + NeuGcNeuAcHex5HexNAc4dHex2 NeuGc2NeuAcHex5HexNAc42544 + + + + + + NeuGc2Hex5HexNAc4dHex2 and/or 2545 + + +NeuGcNeuAcHex6HexNAc4dHex NeuGc3Hex5HexNAc4 2560 + + + +NeuGc2Hex6HexNAc4dHex 2561 + NeuAc2Hex5HexNAc5dHex 2570 + + + + + + + +NeuAcHex5HexNAc5dHex3 2571 + + + + + + + + NeuAc2Hex6HexNAc52586 + + + + + + + + + + + NeuAcHex6HexNAc5dHex22587 + + + + + + + + + + + + Hex7HexNAc6dHexSP 2595 +NeuGcNeuAcHex6HexNAc5 2602 + + + NeuAcHex7HexNAc5dHex and/or2603 + + + + + + + NeuGcHex6HexNAc5dHex2 NeuAcHex8HexNAc5 and/or2619 + + + NeuGcHex7HexNAc5dHex NeuAc2Hex5HexNAc6 2627 +NeuGcHex8HexNAc5 and/or 2635 + + NeuAcHex4HexNAc5dHex4SPNeuAcHex6HexNAc6dHex 2644 + + + + + + + + + + NeuAc2Hex5HexNAc4dHex32659 + + NeuAcHex7HexNAc6 2660 + + + + + + + + + +NeuGcNeuAc2Hex5HexNAc4dHex 2674 + + and/or NeuAc3Hex6HexNAc4NeuAc2Hex4HexNAc5dHex2SP2 2714 + + + + NeuAcHex4HexNAc5dHex4SP2 and/or2715 + + NeuAc3Hex5HexNAc5 NeuAc2Hex5HexNAc5dHex2 2716 +NeuAc2Hex6HexNAc5dHex 2732 + + + + + + + + + + + + +NeuAcHex6HexNAc5dHex3 2733 + + + + + + + + + + + + +NeuGcNeuAcHex6HexNAc5dHex 2748 + NeuAcHex8HexNAc5dHex 2765 +NeuGcHex8HexNAc5dHex and/or 2781 + NeuAcHex9HexNAc5NeuAcHex6HexNAc6dHex2 2791 + + + + Hex6HexNAc6dHex3SP2 2805 +NeuAcHex7HexNAc6dHex 2807 + + + + + + + + + + + + +NeuAc2Hex6HexNAc5dHexSP 2812 + + + + + NeuAcHex6HexNAc5dHex3SP 2813 +NeuGcNeuAc3Hex5HexNAc4 2819 + NeuAc3Hex6HexNAc4dHex and/or 2820 +NeuGcNeuAc2Hex5HexNAc4dHex2 NeuAc3Hex6HexNAc52878 + + + + + + + + + + + + NeuAc2Hex6HexNAc5dHex22879 + + + + + + + + + + + + + NeuAcHex6HexNAc5dHex4 2880 + + + + +NeuGcNeuAc2Hex6HexNAc5 2894 + + NeuAc2Hex7HexNAc5dHex and/or 2895 + +NeuGcNeuAcHex6HexNAc5dHex2 NeuAc3Hex6HexNAc4dHexSP and/or 2900 +NeuGcNeuAc2Hex5HexNAc4dHex2SP NeuGc2Hex6HexNAc5dHex2 2911 +NeuAc2Hex5HexNAc6dHex2 2920 + NeuGc3Hex6HexNAc5 2925 +NeuGcNeuAc2Hex5HexNAc6 2935 + NeuAc2Hex6HexNAc6dHex and/or2936 + + + + + + + NeuGcNeuAcHex5HexNAc6dHex2 NeuAcHex6HexNAc6dHex32937 + + NeuGc2NeuAcHex5HexNAc6 and/or 2951 + NeuAc3Hex5HexNAc4dHex3NeuAc2Hex7HexNAc6 2952 + + + + + + NeuAcHex7HexNAc6dHex22953 + + + + + + + + Hex8HexNAc7dHexSP 2961 + NeuAc2Hex4HexNAc7dHex22961 + NeuAcHex7HexNAc7dHex 3010 + + + NeuAc3Hex6HexNAc5dHex3024 + + + + + + + + + + + + NeuAc2Hex6HexNAc5dHex33025 + + + + + + + + + + + NeuAcHex8HexNAc7 3026 + + + + + +NeuGc3Hex6HexNAc5dHex and/or 3072 + NeuGc2NeuAcHex7HexNAc5NeuAc2Hex6HexNAc6dHex2 3082 + NeuAc2Hex7HexNAc6dHex3098 + + + + + + + + + + + + + NeuAcHex7HexNAc6dHex33099 + + + + + + + + + + + + NeuAc3Hex6HexNAc5dHexSP 3104 + +NeuAc2Hex6HexNAc5dHex3SP 3105 + + NeuAc3Hex6HexNAc5dHex2 3170 + +NeuAc2Hex6HexNAc5dHex4 3171 + + + + + + NeuAcHex8HexNAc7dHex3172 + + + + + + + + + + + NeuAc3Hex6HexNAc6dHex 3227 + +NeuAc2Hex6HexNAc6dHex3 3228 + NeuAc3Hex7HexNAc6 3243 + + +NeuAc2Hex7HexNAc6dHex2 3244 + + + + + NeuAcHex7HexNAc6dHex43245 + + + + + + NeuAc2Hex7HexNAc7dHex 3301 + NeuAcHex7HexNAc7dHex33302 + NeuAc2Hex8HexNAc7 3317 + + + + NeuAcHex8HexNAc7dHex2 3318 + + +NeuAc3Hex7HexNAc6dHex 3389 + + + + + + + NeuAc2Hex7HexNAc6dHex33390 + + + + + + + + + + NeuAcHex7HexNAc6dHex5 and/or 3391 + + +NeuAcHex9HexNAc8 NeuAc2Hex8HexNAc7dHex 3463 + + + + + + + + +NeuAcHex8HexNAc7dHex3 3464 + + + + + + NeuAc2Hex7HexNAc6dHex43536 + + + + + + NeuAcHex9HexNAc8dHex 3537 + + + + + NeuAc3Hex8HexNAc73608 + + NeuAc2Hex8HexNac7dHex2 3609 + + + NeuAcHex8HexNac7dHex43610 + + + + NeuAc4Hex7HexNAc6dHex 3680 + + + NeuAc3Hex7HexNAc6dHex33681 + + + + + + + NeuAc2Hex9HexNAc8 3682 + + + NeuAcHex9HexNAc8dHex23683 + + + NeuAc3Hex8HexNAc7dHex 3754 + + + + NeuAc2Hex8HexNAc7dHex33755 + + + + + + NeuAcHex10HexNAc9 and/or 3756 + + + +NeuAcHex8HexNAc7dHex5 NeuAc4Hex6HexNAc8 3778 + NeuAc3Hex7HexNAc6dHex43827 + + NeuAc2Hex9HexNAc8dHex 3828 + + + + NeuAcHex9HexNAc8dHex33829 + + + + NeuAc2Hex8HexNAc7dHex4 3901 + + + NeuAc2Hex9HexNAc8dHex23974 + + NeuAcHex9HexNAc8dHex4 3975 + + NeuAc4Hex8HexNAc7dHex 4045 +NeuAc3Hex8HexNAc7dHex3 4046 + + NeuAc2Hex10HexNAc9 and/or 4047 + +NeuAc2Hex8HexNAc7dHex5 NeuAc3Hex9HexNAc8dHex 4119 +NeuAc2Hex9HexNAc8dHex3 4120 + HexNAc ≧ 3 and dHex ≧ 1 (includingfucosylated N- glycans) Hex3HexNAc3dHexSP 1338 + Hex4HexNAc3dHexSP1500 + + + + + + + + + + NeuAcHex3HexNAc3dHex1549 + + + + + + + + + + + + Hex4HexNAc3dHex2SP 1646 + +Hex5HexNAc3dHexSP 1662 + Hex6HexNAc3SP and/or1678 + + + + + + + + + + + + + NeuAc2Hex2HexNAc3dHexNeuAcHex3HexNAc3dHexSP2 1709 + + NeuAcHex4HexNAc3dHex1711 + + + + + + + + + + + + + + NeuAcHex5HexNAc3 and/or1727 + + + + + + + + + + + + + NeuGcHex4HexNAc3dHexNeuAcHex4HexNAc3dHexSP 1791 + + + + + + Hex5HexNAc3dHex2SP 1808 +NeuAc2Hex3HexNAc3dHex 1840 + + + + + + + NeuAcHex4HexNAc3dHex2 1857 + +NeuAcHex5HexNAc3dHex and/or 1873 + + + + + + + + + + + + + +NeuGcHex4HexNAc3dHex2 Hex8HexNAc3SP and/or 2002 + + + + + + + + + +NeuAc2Hex4HexNAc3dHex NeuAcHex4HexNAc3dHex3 2003 + + NeuAc2Hex5HexNAc3and/or 2018 + + + + + + + NeuGcNeuAcHex4HexNAc3dHexNeuAcHex5HexNAc3dHex2 2019 + + + NeuGcNeuAcHex5HexNAc3 and/or 2034 +NeuGc2Hex4HexNAc3dHex NeuAcHex6HexNAc3dHex 2035 + + + + + + + + + +NeuAc2Hex4HexNAc3dHexSP and/or 2082 + + + Hex8HexNAc3SP2NeuAcHex6HexNAc3dHexSP 2115 + Hex8HexNAc3dHexSP and/or 2148 +NeuAc2Hex4HexNAc3dHex2 NeuAcHex8HexNAc3SP and/or 2293 +NeuAc3Hex4HexNAc3dHex NeuAc2Hex5HexNAc3dHex2 and/or 2310 +NeuGcNeuAcHex4HexNAc3dHex3 NeuAc2Hex5HexNAc3dHex2SP2390 + + + + + + + + + + NeuAc2Hex6HexNAc3dHexSP 2406 + + +NeuAcHex8HexNAc3dHexSP and/or 2439 + NeuAc3Hex4HexNAc3dHex2NeuAcHex9HexNAc3dHex 2521 + Hex4HexNAc4dHexSP 1703 + + +NeuAcHex3HexNAc4dHex 1752 + Hex5HexNAc4dHexSP 1865 + + + + + + + + + + +Hex4HexNAc5dHexSP 1906 + + NeuAcHex4HexNAc4dHex1914 + + + + + + + + + + + + + Hex5HexNAc4dHex2SP 2011 +Hex6HexNAc4dHexSP 2027 + + Hex7HexNAc4SP and/or 2043 + Hex4HexNAc6SP2and/or NeuAc2Hex3HexNAc4dHex Hex4HexNAc5dHex2SP 2052 + + + +NeuAcHex4HexNAc4dHex2 2060 + + + + + + NeuAcHex4HexNAc4dHexSP2 2074 + +NeuAcHex5HexNAc4dHex 2076 + + + + + + + + + + + + + + NeuAcHex6HexNAc4and/or 2092 + + + + + + + + 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NeuAcHex7HexNAc3dHex3 and/or 2489 + +NeuAcHex4HexNAc5dHex3SP Hex6HexNAc7SP 2490 + NeuAcHex6HexNAc5dHexSPand/or 2521 + + + + NeuAcHex9HexNAc3dHex and/or NeuAc3Hex2HexNAc5dHex2Hex6HexNAc5dHex3SP 2522 + + Hex7HexNAc6dHexSP 2595 + NeuGcHex8HexNAc5and/or 2635 + + NeuAcHex4HexNAc5dHex4SP NeuAc2Hex4HexNAc5dHex2SP22714 + + + + NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + + NeuAc3Hex5HexNAc5NeuAc3Hex5HexNAc4dHex2 and/or 2804 + + NeuAcHex6HexNAc6dHexSP2Hex6HexNAc6dHex3SP2 2805 + NeuAc2Hex6HexNAc5dHexSP 2812 + + + + +NeuAcHex6HexNAc5dHex3SP 2813 + NeuAc3Hex6HexNAc4dHexSP and/or 2900 +NeuGcNeuAc2Hex5HexNAc4dHex2SP NeuAc3Hex6HexNAc5dHexSP 3104 + +NeuAc2Hex6HexNAc5dHex3SP 3105 + + hESC, human embryonic stem cells; EB,embryoid bodies derived from hESC; st.3, stage 3 differentiated cellsderived from hESC; hEF, human fibroblast feeder cells; mEF, murinefibroblast feeder cells; BM MSC, bone-marrow derived mesenchymal stemcells; OB, Osteoblast-differentiated cells derived from BM MSC; CB MSC,cord blood derived mesenchymal stem cells; OB, adipocyte-differentiatedcells derived from CB MSC; CB MNC, cord blood mononuclear cells; CD34+,CD133+, LIN−, and CD8−: subpopulations of CB MNC.

TABLE 48 Neutral N-glycan fraction Sialylated N-glycan fraction (FIG.1.A) (FIG. 1.B) FIG. m/z Composition ST FIG. m/z Composition ST 609609.21 H1N2 M 1565 1565.55 S1H4N3 O 771 771.26 H2N2 M 1678 1678.60S2H2N3F1 O 917 917.32 H2N2F1 M 1711 1711.61 S1H4N3F1 H 933 933.31 H3N2 M1727 1727.60 S1H5N3 H 1079 1079.38 H3N2F1 M 1768 1768.57 S1H4N4 C 10951095.37 H4N2 M 1799 1799.62 S2H4N2F1 O 1120 1120.40 H2N3F1 H 18401840.65 S2H3N3F1 H 1136 1136.40 H3N3 H 1873 1873.66 S1H5N3F1 H 12411241.43 H4N2F1 M 1889 1889.65 S1H6N3 H 1257 1257.42 H5N2 M 1914 1914.68S1H4N4F1 C 1282 1282.45 H3N3F1 H 1930 1930.68 S1H5N4 C 1298 1298.45 H4N3H 1946 1946.67 G1H5N4 C 1323 1323.48 H2N4F1 C 1971 1971.71 S1H4N5 C 13391339.48 H3N4 C 2002 2002.70 S2H4N3F1 H 1403 1403.48 H5N2F1 M 20352035.71 S1H6N3F1 H 1419 1419.48 H6N2 M 2076 2076.74 S1H5N4F1 C 14441444.51 H4N3F1 H 2092 2092.73 G1H5N4F1 C 1460 1460.50 H5N3 H 21172117.76 S1H4N5F1 C 1485 1485.53 H3N4F1 C 2133 2133.76 S1H5N5 C 15011501.53 H4N4 C 2164 2164.75 S2H5N3F1 H 1542 1542.56 H3N5 C 2221 2221.78S2H5N4 C 1565 1565.53 H6N2F1 M 2222 2222.80 S1H5N4F2 C 1581 1581.53 H7N2M 2237 2237.77 G1S1H5N4 C 1590 1590.57 H4N3F2 H 2238 2238.79 S1H6N4F1 C1606 1606.56 H5N3F1 H 2253 2253.76 G2H5N4 C 1622 1622.56 H6N3 H 22632263.82 S1H4N5F2 C 1647 1647.59 H4N4F1 C 2279 2279.82 S1H5N5F1 C 16631663.58 H5N4 C 2295 2295.81 S1H6N5 C 1688 1688.61 H3N5F1 C 2367 2367.83S2H5N4F1 C 1704 1704.61 H4N5 C 2368 2368.85 S1H5N4F3 C 1743 1743.58 H8N2M 2383 2383.83 S2H6N4 C 1768 1768.61 H6N3F1 H 2384 2384.85 S1H6N4F2 C1793 1793.64 H4N4F2 C 2408 2408.86 S2H4N5F1 C 1809 1809.64 H5N4F1 C 24252425.87 S1H5N5F2 C 1825 1825.63 H6N4 C 2441 2441.87 S1H6N5F1 C 18501850.67 H4N5F3 C 2482 2482.90 S1H5N6F2 C 1866 1866.66 H5N5 C 25702570.91 S2H5N5F1 C 1905 1905.63 H9N2 M 2571 2571.93 S1H5N5F3 C 19551955.70 H5N4F2 C 2587 2587.93 S1H6N5F2 C 1987 1987.69 H7N4 C 26032603.92 S1H7N5F1 C 1996 1996.72 H4N5F2 C 2644 2644.95 S1H6N6F1 C 20122012.72 H5N5F1 C 2732 2732.97 S2H6N5F1 C 2028 2028.71 H6N5 C 27332733.99 S1H6N5F3 C 2067 2067.69 H10N2 M 2807 2807.00 S1H7N6F1 C 21012101.76 H5N4F3 C 2878 2878.00 S3H6N5 C 2142 2142.78 H4N5F3 C 28792879.02 S2H6N5F2 C 2174 2174.77 H6N5F1 C 2953 2953.06 S1H7N6F2 C 22292229.74 H11N2 M 3098 3098.10 S2H7N6F1 C 2304 2304.84 H5N5F3 C 30993099.12 S1H7N6F3 C 2361 2361.87 H5N6F2 C 3172 3172.13 S1H8N7F1 C m/z:neutral = [M + Na]⁺, sialylated = [M − H]⁻; Composition: S = NeuAc, G =NeuGc, H = Hex, N = HexNAc, F = dHex; ST (structure class): M =mannose-type, H = hybrid-type, C = complex-type, O = other.

TABLE 49 Comparison of lectin ligand profile in hESCs and MEFs LectinhESC MEF PSA − + MAA + − PNA + − RCA + + + present in cell surface − notpresent in cell surface

TABLE 50 Summary of the results of BM MSC grown on different immobilizedlectin surfaces. Proliferation Effect vs. Coating factor plastic plastic3.8 RCA 1.0 n.g. PSA 3.9 (+) LTA 4.0 + SNA 3.7 (−) GS II 4.9 + UEA 2.1 −ECA 4.4 + MAA 3.7 (−) STA 3.1 − PWA 4.2 + WFA 2.9 − NPA 3.6 (−)Proliferation factor = the number of cells on day 3/the number of cellson day 1. Triplicates were used in calculations. Effect vs. plastic:‘n.g.’ = no growth; ‘−’ = slower growth rate; ‘+’ = faster growth ratethan on plastic; ‘( )’ nearly equal to plastic.

TABLE 51 Detected N-linked and soluble glycome structural typedistribution in stem cells. The column ‘All’ includes all CB stem cellpopulations. hESC MSC All Glycan feature Proposed structure Proportion,% Proportion, % Proportion, % Neutral N-glycan structural features:Hex₅₋₁₀HexNAc₂ High-mannose type/Glc₁ 50-90  30-80  30-90 Hex₁₋₄HexNAc₂dHex₀₋₁ Low-mannose type 5-20 5-20 5-50 n_(HexNAc) = 3 jan_(Hex) ≧ 2 Hybrid-type/Monoantennary 1-20 5-20 1-20 n_(HexNAc) ≧ 4 jan_(Hex) ≧ 2 Complex-type 1-10 5-40 1-40 Hex₁₋₉HexNAc₁ Soluble 1-20 1-301-30 n_(dHex) ≧ 1 Fucosylation 5-20 10-40  5-40 n_(dHex) ≧ 2α2/3/4-linked Fuc 0-5  1-5  0-5  n_(HexNAc) > n_(Hex) ≧ 2 TerminalHexNAc (N > H) 0-20 0-5  0-20 n_(HexNAc) = n_(Hex) ≧ 5 Terminal HexNAc(N = H) 0-10 0-2  0-10 Acidic N-glycan structural features: n_(HexNAc) =3 ja n_(Hex) ≧ 3 Hybrid-type/Monoantennary 1-25 2-20 1-25 n_(HexNAc) ≧ 4ja n_(Hex) ≧ 3 Complex-type 70-99  70-95  70-99  n_(dHex) ≧ 1Fucosylation 60-99  50-80  50-99  n_(dHex) ≧ 2 α2/3/4-linked Fuc 1-401-20 1-40 n_(HexNAc) > n_(Hex) ≧ 2 Terminal HexNAc (N > H) 1-25 0-5 0-25 n_(HexNAc) = n_(Hex) ≧ 5 Terminal HexNAc (N = H) 1-30 0-5  0-30 +80Da Sulphate or phosphate ester 0-50 0-40 0-50

TABLE 52 Neutral glycan signals of human stem cell glycosphingolipidglycans. Proposed composition m/z Hex2dHex 511.24 511 Hex3 527.15 527Hex2HexNAc 568.19 568 Hex2HexNAcdHex 714.24 714 Hex3HexNAc 730.24 730Hex2HexNAc2 771.26 771 HexHexNAc3 812.29 812 Hex3HexNAcdHex 876.30 876Hex4HexNAc 892.29 892 HexHexNAc2dHex2 901.33 901 Hex2HexNAc2dHex 917.32917 Hex3HexNAc2 933.31 933 Hex2HexNAc3 974.34 974 Hex2HexNAcdHex31006.36 1006 Hex3HexNAcdHex2 1022.35 1022 Hex5HexNAc 1054.34 1054Hex2HexNAc2dHex2 1063.38 1063 Hex2HexNAc2dHex 1079.38 1079 Hex4HexNAc21095.37 1095 Hex3HexNAc3 1136.40 1136 Hex6HexNAc 1216.40 1216Hex3HexNAc2dHex2 1225.43 1225 Hex4HexNAc2dHex 1241.43 1241 Hex5HexNAc21257.42 1257 Hex3HexNAc3dHex 1282.45 1282 Hex4HexNAc3 1298.45 1298Hex2HexNAc4dHex 1323.48 1323 Hex3HexNAc2dHex3 1371.49 1371 Hex7HexNAc1378.45 1378 Hex4HexNAc2dHex2 1387.49 1387 Hex5HexNAc2dHex 1403.48 1403Hex6HexNAc2 1419.48 1419 Hex3HexNAc3dHex2 1428.51 1428 Hex4HexNAc3dHex1444.51 1444 Hex5HexNAc3 1460.50 1460 Hex4HexNAc2dHex3 1533.54 1533Hex8HexNAc 1540.5 1540 Hex6HexNAc2dHex 1565.53 1565 Hex4HexNAc3dHex21590.57 1590 Hex5HexNAc3dHex 1606.56 1606 Hex6HexNAc3 1622.56 1622Hex9HexNAc 1702.56 1702 Hex4HexNAc3dHex3 1736.62 1736 Hex5HexNAc3dHex21752.62 1752 Hex4HexNAc5dHex 1850.67 1850 Hex10HexNAc 1864.61 1864Hex7HexNAc2dHex2 1873.64 1873 Hex4HexNAc3dHex4 1882.68 1882Hex5HexNAc3dHex3 1898.68 1898 Hex5HexNAc4dHex2 1955.70 1955 Hex11HexNAc2026.66 2026 Hex5HexNAc4dHex3 2101.76 2101 Hex6HexNAc4dHex2 2117.75 2117Hex4HexNAc5dHex3 2142.78 2142 Hex12HexNAc 2188.71 2188

TABLE 53 Acidic glycan signals of human stem cell glycosphingolipidglycans. Proposed composition m/z NeuAcHexHexNAcdHex 819.29 819NeuAcHex2HexNAc 835.28 835 NeuAc2Hex2 905.30 905 NeuAcHexHexNAcdHex2965.35 965 NeuAcHex3HexNAc 997.34 997 NeuAc2Hex2HexNAc 1126.38 1126NeuAcHex3HexNAcdHex 1143.39 1143 Hex4HexNAc2SP 1151.33 1151NeuAcHex4HexNAc 1159.39 1159 NeuAcHexHexNAc2dHex2 1168.43 1168NeuAcHex3HexNAc2 1200.42 1200 NeuGcHex3HexNAc2 1216.41 1216Hex2HexNAc4SP 1233.38 1233 NeuAc2Hex3HexNAc 1288.43 1288NeuAc2HexHexNAc2dHex 1313.46 1313 NeuAcHex2HexNAc2dHex2 1330.48 1330NeuAcHex4HexNAc2 1362.47 1362 NeuAc2Hex4HexNAc/ 1450.48 1450NeuAc2HexHexNAc3SP NeuAcHex4HexNAc2dHex 1508.53 1508NeuAcHex2HexNAc3dHex2 1533.56 1533 Hex6HexNAc2SP2/ 1555.47/1555.39 1555NeuAc2Hex2HexNac2dHexSP NeuAcHex4HexNAc3 1565.55 1565 NeuAcHex5HexNAc31727.60 1727 NeuGcHex5HexNAc3 1743.60 1743 NeuAcHex5HexNAc3dHex 1873.661873 NeuAcHex6HexNAc3 1889.65 1889 NeuAcHex3HexNAc4dHex2 1898.69 1898NeuAc2Hex3HexNac3dHexSP 1920.60 1920 NeuAc2Hex5HexNAc3 2018.70 2018NeuAcHex6HexNAc3dHex 2035.71 2035 NeuAcHex6HexNAc4 2092.73 2092NeuGcHex6HexNAc4 2108.73 2108 NeuAcHex4HexNAc4dHex3SP 2286.76 2286NeuAc2Hex5HexNAc4SP 2301.73 2301 NeuGc3Hex4HexNAc4 2398.80 2398NeuAcHex5HexNAc4dHex3SP/ 2448.81 2448 NeuAcHex8HexNAc2dHex3Hex7HexNAc6SP 2449.81 2449 NeuGc2Hex7HexNAc5 2780.95 2780NeuGcHex8HexNAc5dHex/ 2781.97 2781 NeuAcHex9HexNAc5

REFERENCES

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1. A method for the analysis of the status of stem cells and/or formanipulation of stem cells comprising a step of detecting a glycanstructure from a sample of stem cells, wherein said glycan structure isselected from the group consisting of: a terminal Galβ3; a terminalmannose; a terminal SAα3Galβ3, wherein SA is a sialic acid; a terminalGalβ4; and a terminal SAα3Galβ4, wherein SA is a sialic acid, with theprovisions that a) the stem cells are not cells of a cancer cell lineand b) when the cells are embryonal stem cells, the glycan structure isnot a SSEA structure, or the molar proportions of SSEA-3 and SSEA-4structures are characterized or c) when the stem cells are not CD34+hematopoietic stem cells the structure is not SAα3Galβ4.
 2. A method forthe analysis of the status of stem cells and/or manipulation of stemcells by detecting a glycan structure with a terminal Galβ3 from asample of stem cells with the provisions that when the cells areembryonal stem cells, the glycan structure is not a SSEA structure. 3.The method according to claim 2, wherein the stem cells are mesenchymalstem cells or hematopoietic stem cells.
 4. The method according to claim2 or 3, wherein said glycan structure is an O-glycan or Galβ3GlcNAccomprising epitope.
 5. The method according to claim 2, wherein thedisaccharide epitope is terminal structure of a complex N-glycan or aneolacto or lacto glycolipid or an O-glycan and or O-glycan corestructure R₁Galβ3{R₃}_(n1)HexNAc, wherein HexNAc is GalNAc or GlcNAc,with the provision that HexNAx can be GalNAc only when the Gal isGalβ3-linked, preferably the terminal structure is β-linked to terminalManα3 and/or Manα6 on a N-glycan core epitope Manα3/6Manβ4GlcNAcXyR₂,and/or to a glycolipid structure Galβ4GlcβCer and/or to O-glycan coreGalβ3GalNAc or it is the O-glycan core.
 6. The method as described inclaim 2, wherein the structure is Galβ3GalNAc and an O-glycan corestructure.
 7. The method as described in claim 5, wherein the HexNac isGlcNAc.
 8. The method according to claim 3 wherein the disaccharideepitope is terminal structure of a complex N-glycan or a lactoglycolipid or a O-glycan R₁Galβ3{R₃}_(n1)GlcNAcn₂, preferably theterminal structure is β-linked to terminal Manα3 and/or Manα6 on aN-glycan core epitope Manα3/6Manβ4GlcNAcXyR₂, and/or to a glycolipidstructure Galβ4GlcβCer and/or to O-glycan core Galβ3GalNAcαSer/Thr. 9.The method according to claim 8, wherein the terminal structure isGalβ3GlcNAc or Galβ3(Fucα4)GlcNAc.
 10. The method according to any oneof claims 8-9, wherein the structures can be recognized from byβ3-galactosidase.
 11. The method according to any one of claims 8-10,wherein the structure is N-glycan or O-glycan structureor a glycolipidlinked structure.
 12. The method according to any one of claims 8-11,wherein the structure is quantitatively determined.
 13. The methodaccording to any one of claims 8-12, wherein the amount of cellsanalysed is between 1000 and 10 million.
 14. The method according to anyone of claims 8-13, wherein the detection is performed by a binder beinga protein selected from the group consisting of monoclonal antibody,glycosidase, glycosyl transferring enzyme, plant lectin, animal lectinor a peptide mimetic thereof.
 15. The method according to any one ofclaims 8-14, wherein the detection is performed by mass spectrometryfrom a stem cell glycome produced by the method for production ofglycome compositions according to any of the previous claims comprisingsteps of 1) releasing non-derivatized glycome composition from stemcells 2) purifying the glycome composition by microchromatographymethods involving use of hydrophophic and hydrophilic chromatography andoptionally anion exchange chromatography.
 16. A method for the analysisof the status of stem cells and/or for manipulation of stem cellscomprising a step of detecting a glycan structure with a terminalSAα3Galβ3 from a sample of stem cells, wherein SA is a sialic acid, withthe provisions that when the cells are embryonal stem cells, the glycanstructure is not a SSEA structure.
 17. The method according to claim 16,wherein the terminal structure is SAα3Galβ3GlcNAc orSAα3Galβ3(Fucα4)GlcNAc.
 18. The method according to claim 16 or 17,wherein the structure is cleavable by α3-sialidase.
 19. The methodaccording to claim 16 or 17, wherein the method is further defined asdescribed in claims 11 or
 12. 20. The method according to claim 16 or17, wherein the method is further defined as described in any of theclaims 13-15.
 21. A method for the analysis of the status of stem cellsand/or for manipulation of stem cells comprising a step of detecting aglycan structure with a terminal Galβ4 from a sample of stem cells. 22.The method according to claim 21, wherein the terminal structure isGalβ4GlcNAc or Galβ4(Fucα3)GlcNAc.
 23. The method according to any ofclaims 21 or 22, wherein the structures can be recognized byβ4-galactosidase.
 24. The method according to any of the claims claims21-23, wherein the structure is protein linked N-glycan or O-glycanstructure or glycolipid.
 25. The method according to any one of claims21-24, wherein the structure is quantitatively determined.
 26. Themethod according to any one of claims 21-25, wherein the amount of cellsanalysed is between 1000 and 10 million.
 27. The method according to anyone of claims 21-26, wherein the detection is performed by a binderbeing a protein selected from the group consisting of monoclonalantibody, glycosidase, glycosyl transferring enzyme, plant lectin,animal lectin or a peptide mimetic thereof.
 28. The method according toany one of claims 21-27, wherein the detection is performed by massspectrometry from a stem cell glycome produced by the method forproduction of glycome compositions according to any of the previousclaims comprising steps of 1) releasing non-derivatized glycomecomposition from stem cells 2) purifying the glycome compositionmicrochromatography methods involving use of hydrophophic andhydrophilic chromatography and optionally anion exchange chromatography.29. A method for the analysis of the status of stem cells and/or formanipulation of stem cells comprising a step of detecting a glycanstructure with a terminal SAα3Galβ4 from a sample of stem cells, whereinSA is a sialic acid, with the provision that the stem cells are notCD34+ hematopoietic stem cells.
 30. The method according to the claim29, wherein the structure is cleavable by α3-sialidase.
 31. The methodaccording to the claim 29 or 30, wherein the method is further definedas described in claims 11 or
 12. 32. The method according to the claim29 or 30, wherein the method is further defined as described in any ofthe claims 13-15.
 33. The method according to the claim 1, wherein thedisaccharide epitope is Manβ4GlcNAc structure in the core structure ofN-linked glycan according to the Formula:[Manα3]_(n1)(Manα6)_(n2)Manβ4GlcNAcβ4(Fucα6)₀₋₁YxR₂, wherein n1 and n2are integers 0 or 1, independently indicating the presence or absence ofthe terminal Man-residue, and wherein the non-reducing end terminalManα3/Manα6-residues can be elongated to the complex type, especiallybiantennary structures or to mannose type (high-Man and/or low Man) orto hybrid type structures for the analysis of the status of stem cellsand/or manipulation of the stem cells.
 34. The method according to theclaim 6, wherein the Manβ4GlcNAc-epitope is essentially devoid ofadditional GlcNAc-substitutions, preferably the amount of the GlcNAcsubstitution is less than 8%.
 35. A marker structure compositioncomprising the core structure as described in claim 6, wherein theManβ4GlcNAc-epitope comprises between 1-8% of the GlcNAc substitutions.36. A composition comprising the core structure as described in claim33, wherein the Manβ4GlcNAc-epitope comprise between 1-8% of the GlcNAcsubstitutions.
 37. A low mannose type glycan marker structure accordingto claim 33, wherein the structure of the marker glycan is according tothe Formula (Manα)₁₋₃Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAc and the terminalManα-residues are devoid of substitutions by other monosaccharideresidues.
 38. A low mannose type glycan marker structure according toclaim 37, wherein the structure of the marker glycan is according to theFormula[Mα3]_(n2){[Mα6)]_(n4)}[Mα6]_(n5){[Mα3]_(n8)}Mβ4GNβ4[{Fucα6}]_(m)GNyR₂wherein p, n2, n4, n5, n8, and m are either independently 0 or 1; withthe proviso that when n2 is 0, also n1 is 0; when n4 is 0, also n3 is 0;when n5 is 0, also n1, n2, n3, and n4 are 0; when n7 is 0, also n6 is 0;when n8 is 0, also n6 and n7 are 0; the sum of n1, n2, n3, n4, n5, n6,n7, and n8 is less than or equal to (m+3); [ ] indicates determinanteither being present or absent depending on the value of n2, n4, n5, n8,and m; and { } indicates a branch in the structure.
 39. The markerstructure according to the claim 38, wherein the structure of the markerglycan is according to the Formula(Manα)₀₋₁Manα6(Manα3)Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAc
 40. A low mannosetype glycan marker structure according to claim 33, wherein thestructure of the marker glycan is according to the Formula(Manα6)₀₋₁(Manα3)₀₋₁Manβ4GlcNAcβ4(Fucα6)₀₋₁GlcNAc, when either of theMana-residues is present or absent.
 41. A glycan composition comprising1-40% of total glycome of the glycan structures described in claim 33.42. The glycan composition according to the claim 41, comprisingstructures according to the claim
 40. 43. A high-mannose type glycanmarker structure according to claim 33, wherein the structure of themarker glycan is according to the Formula:[Mα2]_(n1)[Mα3]_(n2){[Mα2]_(n3)[Mα6)]_(n4)}[Mα6]_(n5){[Mα2]_(n6)[Mα2]_(n7)[Mα3]_(n8)}Mβ4GNβ4GNyR₂wherein n1, n2, n3, n4, n5, n6, n7, and n8 are either independently 0 or1; with the proviso that when n2 is 0, also n1 is 0; when n4 is 0, alson3 is 0; when n5 is 0, also n1, n2, n3, and n4 are 0; when n7 is 0, alson6 is 0; when n8 is 0, also n6 and n7 are 0; and the sum of n1, n2, n3,n4, n5, n6, n7, and n8 is an integer from 4 to 8; y is anomeric linkagestructure α and/or β or linkage from derivatized anomeric carbon, and R₂is reducing end hydroxyl, chemical reducing end derivative or naturalasparagine N-glycoside derivative such as asparagine N-glycosidesincluding asparagines N-glycoside aminoacid and/or peptides derived fromprotein; [ ] indicates determinant either being present or absentdepending on the value of n1, n2, n3, n4, n5, n6, n7, and n8; and { }indicates a branch in the structure.
 44. The high-mannose type glycanmarker structure according to claim 43, wherein all n1, n2, n3, n4, n5,n6, n7, and n8 are
 1. 45. A neutral glycan composition comprising about5-50% low-mannose type glycans, and optionally 30-90% high-mannose typeglycans, and/or 1-20% hybrid-type or monoantennary glycans, and/or 1-40%complex-type glycans.
 46. A method for the analysis of the status ofstem cells and/or for manipulation of stem cells comprising a step ofdetecting a glycan structure with a terminal mannose from a sample ofstem cells.
 47. The method according to claim 46, wherein the structureis cleavable by α-mannosidase and a glycan marker or composition is asdescribed in any of the claims 37-45.
 48. The method according to claim46 or 47, wherein the method is further defined as described in claims11 or
 12. 49. The method according to claim 46 or 47, wherein the methodis further defined as described in any of the claims 13-15.
 50. Themethod according to claim 1, wherein the detection comprises one or moreof the following methods: i. preparation of substrate cell materials foranalysis by the use of a chemical buffer solution, or by the use ofdetergents, chemical reagents and/or enzymes; ii. release of glycome(s)from the cells, including various subglycome types based on glycan core,charge and other structural features, by the use of reagents, thecarbohydrate content of which is controlled; iii. purification ofglycomes and various subglycomes from complex mixtures; iv. preferredglycome analysis, including profiling methods such as mass spectrometryand/or NMR spectroscopy; v. The data processing and analysis, especiallycomparative methods between different sample types and quantitativeanalysis of glycome data obtained.
 51. The method according to claim 50,wherein the glycome is non-derivatized or singly derivatized, preferablyreducing end derivatized oligosaccharide composition.
 52. The methodaccording to claim 50, wherein the glycome is non-derivatizedoligosaccharide composition.
 53. The method according to claim 50,wherein the glycome comprises oligosaccharides with molecular weightfrom about 400 to about 4000, preferably from about 600 to about 3500.54. The method according to claim 50, wherein the amount of cells to beanalysed is between 10³ and 10⁶ cells.
 55. The method according to claim50, wherein the glycan structure is a N-glycan subglycome comprisingN-Glycans with N-glycan core structure and said N-Glycans beingreleasable from cells by N-glycosidase.
 56. The method according toclaim 55, wherein the N-glycan core structure isManβ4GlcNAcβ4(Fucα6)_(n)GlcNAc, wherein n is 0 or
 1. 57. The methodaccording to claim 50, wherein the group of glycan structures comprisesoligosaccharides in specific amounts shown in Tables and Figures of thespecification.
 58. The method according to claim 50, wherein the glycansare released from the surface of the cells.
 59. The method according toclaim 50, wherein the stem cell preparation comprises human early bloodcells or mesenchymal cells derived thereof
 60. The method according toclaim 50, wherein said cell preparation comprises a cord blood cellpopulation.
 61. The method according to claim 50, wherein said cellpreparation comprises embryonal-type cell population.
 62. The methodaccording to claim 50 for evaluating the status of an isolated earlyhuman cell population.
 63. The method according to claim 50 forevaluating the differentiation status of an isolated early human cellpopulation.
 64. The method according to claim 50, wherein said cellpreparation comprises human early blood cells or mesenchymal cellsderived thereof.
 65. The method according to claim 50, wherein said cellpreparation comprises a embryonal-type cell population.
 66. The methodaccording to claim 50, wherein said cell preparation comprises a cordblood cell population.
 67. The method according to claim 50, wherein thepresence or absence of cell surface glycomes of said cell preparation isdetected.
 68. The method according to claim 50, wherein said cellpreparation is evaluated with regard to a contaminating structure in acell population of said cell preparation or a change in the status ofthe cell population.
 69. The method according to claim 50 for thecontrol of cell status and/or potential contaminations by physicaland/chemical means preferably by glycosylation analysis using massspectrometric analysis of glycans in said cell preparation.
 70. Themethod according to claim 50 for the control of a variation in rawmaterial cell population.
 71. The method according to claim 70, whereinone specific variation is detected.
 72. The method according to claim50, wherein the cell status is controlled during cell culture or duringcell purification, in context with cell storage or handling at lowertemperatures, or in context with cryopreservation of cells.
 73. Themethod according to claim 72, wherein time dependent changes of cellstatus are detected.
 74. The method according to claim 73, wherein timedependent changes of cell status depend on the nutritional status of thecells, confluency of the cell culture, density of the cells, changes ingenetic stability of the cells, integrity of the cell structures or cellage, or chemical, physical, or biochemical factors affecting the cells.75. The method according to claim 50 for evaluating the malignancy of anisolated early human cell population.
 76. The method according to claim50, wherein said method comprises the steps of: i) preparing a stem cellsample containing glycans for the analysis; ii) releasing total glycansor total glycan groups from the stem cell sample, or extracting freeglycans from the stem cell sample; iii) optionally modifying glycans;iv) purifing the glycan fraction/fractions from biological material ofthe sample; v) optionally modifying glycans and/or producing a glycomeMALDi-matrix compostion for mass psectormetric analysis vi) analysingthe composition of the released glycans by mass spectrometry; vii)optionally presenting the data about released glycans quantitatively andcomparing the quantitative data set with another data set from anotherstem cell sample; viii) comparing data about the released glycansquantitatively or qualitatively with data produced from another stemcell sample, optionally using a glycan score method.
 77. Method formodifying cell surface glycans of an isolated human stem cellpopulation, the method comprising the steps of: a) contacting said cellpopulation with a reagent or enzyme capable of modifying the surfaceglycans of said cell population; b) optionally isolating a modified cellpopulation obtained from step a).
 78. An isolated human stem cellpopulation with modified cell surface glycans obtained by the methodaccording to claim
 77. 79. An essentially pure oligosaccharide glycomecomposition of multiple oligosaccharides obtained by the methodaccording to claim
 50. 80. The method according to the claim 50, whereinthe detection is preformed by a binder being a recombinant proteinselected from the group monoclonal antibody, glycosidase, glycosyltransferring enzyme, plant lectin, animal lectin or a peptide mimeticthereof.
 81. The method according to the claim 80, wherein therecombinant protein is a high specificity binder recognizing at leastpartially two monosaccharide structures and bond structure between themonosaccharide residues.
 82. The method according to the claim 80,wherein the binder protein is labelled by a detectable marker structure.83. The method according to the claim 80 or 82, wherein the binder isused for sorting or selecting human stem cells from biological materialsor samples including cell materials comprising other cell types.
 84. Themethod according to the claim 80 or 82, wherein the binder is used forsorting or selecting between different human stem cell types.
 85. A stemcell glycan marker structure compring at least one terminal or coreglycan sequence structure according to the FormulaR₁Hexβz{R₃}_(n1)Hex(NAc)_(n2)XyR₂, wherein X is glycosidically linkeddisaccharide epitope β4(Fucα6)_(n)GN, wherein n is 0 or 1, or X isnothing, or when n2 is 0, X can be βCer, a ceramide or part orderivative thereof and yR₂ is nothing or reducing end glycan core partcomprising a glycolipid, or O-glycan or glycosaminoglycan or N-glycancore structures; Hex is Gal or Man or GlcA; HexNAc is GlcNAc or GalNAc;y is anomeric linkage structure a and/or P or linkage from derivatizedanomeric carbon; z is linkage position 3 or 4, with the provision thatwhen z is 4 then HexNAc is GlcNAc and then Hex is Man or Hex is Gal orHex is GlcA, and when z is 3 then Hex is GlcA or Gal and HexNAc isGlcNAc or GalNAc and with the proviso that Hex can be Man only when n1is 0 and n2 is 1 n1 is 0 or 1 indicating presence or absence of R3; n2is 0 or 1, indicating the presence or absence of NAc, with the provisothat n2 can be 0 only when Hexβz is Galβ4, and n2 is preferably 0, n2 is1—structures are preferably derived from glycolipids; R₁ indicates 1-4,preferably 1-3, natural type carbohydrate substituents linked to thecore structures or nothing; R₂ is a natural O-glycan, N-glycan orglycolipid reducing end structure or a chemical reducing endderivatization structure; R3 is nothing or a branching structurerespesenting a GlcNAcβ6 or an oligosaccharide with GlcNAcβ6 at itsreducing end linked to GalNAc or when Hex is Gal and HexNAc is GlcNActhe then when z is 3 R3 is Fucα4 or nothing and when z is 4 R3 is Fucα3or nothing, for the analysis of the status of stem cells and/ormanipulation of the stem cells.
 86. The method according to claim 85,wherein said glycan structure is disaccharide epitope according to theFormula:R₁Hexβ4{R₃}_(n1)Glc(NAc)_(n2)XyR₂, wherein Hex is Gal or Man.